Дисертації з теми "Empty virus like particles"

The re-design and controlled self-assembly of natural systems into non-natural functional products is a quickly developing area of Synthetic Biology. Specifically, the manipulation of existing, and the introduction of new protein-protein interactions will allow great advances in bionanotechnology. In nature, protein-protein assemblies mediate many cellular processes and exhibit complex and efficient functions. It is thus rational to assume human-guided biomolecular assemblies could embody equally complex functionality designed to address current human needs. Here we present the design and preparation of a Virus-Like Particle (VLP) engineered from the cholera toxin B-subunit (CTB). This was achieved via the de novo design of a protein-protein interface between CTB subunits consisting of coiled-coil C-terminal extensions and modification to the CTB surface. A combination of computational methods was used to suggest mutations which should reduce the ΔΔG of interaction across the interface. CTB is a natural homopentamer with inbuilt cell targeting and endocytic triggering mechanism. Future applications for the VLP could include use as a drug delivery vehicle to transport protected therapeutic agents to targeted cell types. Through our investigations it became apparent that the CTB-VLP structures behaved in a similar manner to naturally occurring virus coat proteins, which suggests the successful biomimicry of these complex systems. This study provides a basis for the development of further VLPs from other homomultimeric proteins, especially further classes of homopentameric bacterial toxins.

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.

Since there is an urgent need for development of vaccines and antiviral agents to combat influenza pandemics, this study aimed to develop influenza virus-like particles (VLPs) and aptamers targeting the virus particles as vaccine and antiviral agent candidates. Influenza VLPs containing three structural proteins of hemagglutinin (HA), neuraminidase (NA) and matrix 1 (M1) derived from influenza A/Hong Kong/01/2009 (H1N1) virus (HK/01) were constructed using a Bac-to-Bac baculovirus expression system. The expressed VLPs were purified by sucrose density gradient ultracentrifugation and characterized by Western blotting analysis and transmission electron microscopy. The immune responses and protective efficacy induced by VLPs were compared with those elicited by the clinically used Panenza vaccine in BALB/c mouse model. The results showed that two-dose vaccination with both VLP and the Panenza vaccine could confer complete protection. Single-dose vaccination with VLP could also provide 100% protection against lethal virus challenge, whereas single dose of an equal amount (based on HA content) of the Panenza vaccination just provided incomplete protection (67% survival rate) against the lethal virus challenge. Compared to the Panenza vaccination, the VLP vaccination could induce higher and broader antibody responses and higher viral specific T help (Th) cell and cytotoxic T lymphocyte (CTL) responses. Notably, a novel finding in this study is that the VLP vaccination could induce antibodies to inhibit virus release from infected MDCK cells, although the underlined mechanism needed to be further studied. These results indicated that influenza VLP might be a more effective and safe vaccine candidate which could be developed into an alternative vaccine for the control of epidemic and pandemic influenza in the future. To develop aptamers as antiviral agents against influenza, I sought to use influenza VLPs as target for ssDNA aptamer selection. After 11 rounds of selection using the systemic evolution of ligandsby exponential enrichment (SELEX),the recovered DNA molecules were PCR-amplified, gel purified and cloned into pCR-Blunt II TOPO vector for sequencing. The sequencing results showed that one aptamer Va-1 was markedly enriched, which was accounted for 59% (13/22) of the selected aptamers. Compared to the other non-enriched aptamers, the enriched aptamer Va-1 showed the highest binding affinity to the UV inactivated influenza HK/01 virus. It was also shown that the aptamer Va-1 specifically bound to the HK/01 stain while it could not bind other respiratory viruses even the PR8 strain within the H1N1 subtype. It was further demonstrated that the aptamer Va-1 could only bind to NA protein in a dose-dependent manner but not bind to HA and M1 proteins. Unfortunately, the selected aptamer did not show any antiviral effects. However, it may be potentially developed into a diagnostic and analytic agent because its binding activity was comparable with that of the commercial anti-NA antibody. In conclusion, the influenza VLPs may be a promising vaccine candidate for the control of influenza virus infection and the selected aptamer may be potentially developed into an alternative tool for influenza virus detection.
published_or_final_version
Microbiology
Doctoral
Doctor of Philosophy

Porcine reproductive and respiratory syndrome (PRRS) is the most significant infectious disease currently affecting the swine industry worldwide. Several inactivated and modified live vaccines (MLV) have been developed to curb PRRSV infections. The unsatisfactory efficacy and safety of these vaccines, drives for the development of new generation PRRS universal vaccines. Virus like particles (VLPs) based vaccines are gaining increasing acceptance compared to subunit vaccines, as they present the antigens in more veritable conformation and are even readily recognized by the immune system. Hepatitis B virus (HBV) core antigen (HBcAg) is very well studied and has been successfully used as a carrier for more than 100 other viral sequences. In this study, hybrid HBcAg VLPs are generated by fusion of the conserved protective epitopes of PRRSV and expressed in E. coli. An optimized purification protocol that overcomes issues from ultracentrifugation is developed to obtain hybrid HBcAg VLP protein from the inclusion bodies. This hybrid HBcAg VLP protein self assembled to 23nm VLPs that were shown to block virus infection of susceptible cells when tested on MARC 145 cells. Therefore, the safety of non-infectious and non-replicable VLPs and production through low-cost E. coli fermentation may make this vaccine competitive against current vaccines on both efficacy and cost.
Master of Science

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.

Monoliths are an alternative stationary phase format to conventional particle based media for large biomolecules. Conventional resins suffer from limited capacities and flow rates when used for viruses, virus-like particles (VLP) and other nanoplex materials. Monoliths provide an open pore structure to improve pressure drops and mass transport via convective flow. The challenging capture of a VLP from clarified yeast homogenate was used to develop a new monolith separation which found hydrophobic interaction based separation using a hydroxyl derivatised monolith had the best performance. The monolith was then compared to a known beaded resin method, where the dynamic binding capacity increased three-fold for the monolith with 90% recovery of the VLP. Confocal microscopy was used to visualise lipid contaminants, deriving from the homogenised yeast. The lipid formed a layer on top of the column, even after column regeneration, resulting in increasing pressure drops over a number of cycles. Removal of 70% of the lipid pre-column by Amberlite/XAD-4 beads significantly reduced the fouling process. Applying a reduced lipid feed versus an untreated feed further increased the dynamic binding capacity of the monolith from 0.11 mg/mL column to 0.25mg/mL column. Control of chromatographic conditions can impact the product concentration during elution. Critical parameters which influenced the concentration of measureable VLP eluted included column contact time, salt concentration in mobile phase, and inclusion of lipid. The parameters were co-dependant with a crude lipid feed loaded at low salt and extra wash time of 40 minutes causing the largest decrease of 40%. Reducing the time of contact between the column and the VLP helped reduce such adverse effects. Increasing the flow rate in the column had no effect on the elution profile with crude or reduced lipid feeds. This informs process development strategies for the future use of monoliths in vaccine bioprocessing.

O vírus Chikungunya (CHIKV) é um alfavírus, transmitido por mosquitos, que causa infecção aguda no Homem caracterizada por febre, mialgia e poliartrite dolorosa e incapacitante que pode durar meses ou anos. Descrito pela primeira vez na Tanzânia em 1952, tornou-se endémico em África, Índia e sudeste Asiático. Desde 2005, os surtos no oceano Índico e no continente Asiático têm atingido proporções e virulência atípicas, com muitos milhares de indivíduos infectados e mortalidade associada. A mobilidade de indivíduos infectados e a alteração dos padrões de distribuição e abundância dos vectores devido a alterações climáticas, tornaram o CHIKV uma ameaça global sem estratégias de controlo eficazes. Uma abordagem para o controlo da transmissão será o desenvolvimento de uma vacina eficaz. As virus-like particles (VLPs) são uma classe segura e eficaz de vacinas que simulam a estrutura nativa das partículas virais. Neste trabalho pretendemos obter um vector de expressão recombinante, capaz de induzir a síntese de VLPs de CHIKV quando transfectado em culturas celulares, como fonte da sequência codificante da poliproteína estrutural para clonagem em baculovírus e produção de VLPs em células de insecto. A ORF estrutural de CHIKV foi amplificada por RT-PCR como um fragmento SacI-NotI e clonada no vector de expressão pLEXm a jusante do promotor forte da beta actina de galinha. Obtiveram-se vários clones com o tamanho correcto do inserto, os quais foram usados para transfectar células HEK 293T usando polietilenimina como agente de fusão. A análise por imunofluorescência indirecta (IF) e Western blotting (WB) de células transfectadas, usando um soro policlonal anti-CHIKV, demonstrou que cinco vectores recombinantes expressavam antigénios virais. Um clone apresentou níveis elevados de fluorescência apenas quando da permeabilização celular e induz, muito provavelmente, a síntese de uma glicoproteína E2 truncada (26 kDa) que não é transportada até à membrana citoplasmática. Três clones deram origem a intensidades de fluorescência relativamente fracas que poderão estar correlacionadas com menor taxa de tradução e/ou processsamento incompleto. As células transfectadas com pLCHIKS67 revelaram fluorescência e padrão proteico em WB semelhante aos de células infectadas com CHIKV. Ainda, o seu sobrenandante de cultura quando precipitado com PEG 8000 revelou (WB) uma composição de antigénios virais idêntica ao precipitado correspondente de células infectadas com CHIKV. A análise por microscopia electrónica de transmissão de células infectadas com CHIKV e de células transfectadas com pLCHIKS67 demonstrou a presença de VLPs nestas últimas, com dimensões e morfologia idênticas às partículas virais, e ainda um extenso rearranjo de membranas intracelulares, observado igualmente nas células infectadas, o qual deverá estar associado à tradução, processamento e transporte das proteínas estruturais. De acordo com os resultados obtidos, pLCHIKS67 será uma boa fonte da sequência nucleotídica da poliproteína estrutural para usar na geração de baculovírus produtores de VPLs de CHIKV.
Chikungunya vírus (CHIKV) is a mosquito-transmitted alphavirus that causes an acute infection in humans, characterized by fever, myalgia and painful invalidating poly-arthralgia that may last for months. CHIKV was first reported in Tanzania in 1952 and became endemic in Africa, India and South-East Asia. Since 2005, outbreaks in the Indian Ocean and Asian continent reached an atypical magnitude and virulence, with many thousands of people infected and associated fatalities. Travelling and changing patterns of vector distribution and abundance due to climatic changes, make CHIKV a global threat without effective control strategies. One approach to reduce the CHIKV burden is the development of a vaccine. Virus-like particles are a safe and highly effective class of recombinant vaccines that mimic the overall structure of virus particles. Accordingly, we aimed to obtain a recombinant vector, able to induce CHIKV VLPs upon cell transfection, as a source of CHIKV structural genes to construct a recombinant baculovirus for VLP production in insect cells. CHIKV structural ORF was amplified by RT-PCR as a SacI-NotI fragment, and cloned into the mammalian expression vector pLEXm downstream from the strong chick beta actin promoter. Several clones with the correct insert size were obtained and transfected into HEK 293T cells using polyethylenimine as the transfection reagent. Five recombinant vectors were shown to express CHIKV proteins by immunofluorescence (IF) and Western blotting (WB) with a polyclonal serum against CHIKV. One clone, with high levels of IF labelling, demonstrated a truncated viral polyprotein of 26 kDa on WB while three others, with a lower IF signal, originated low levels of viral envelope glycoproteins correctly processed. Cells transfected with clone pLCHIKS67 showed IF staining and viral glycoprotein WB pattern similar with those of cells infected with CHIKV. Precipitation with PEG 8000 of clarified cell culture medium from pLCHIKS67 transfected cells and from CHIKV infected cells generated pellets with an identical content of viral glycoproteins on WB, strongly indicating that pLCHIKS67 transfected cells express CHIKV structural proteins that are assembled into VLPs. Transmission electron microscopy of transfected cells demonstrated cytoplasmic membrane rearrangements similar to the vesicle arrays observed in CHIKV infected cells and confirmed the assembly of VLPs with size and morphology similar to the virus particles produced in infected cells. Therefore, pLCHIKS67 will be used as a source of CHIKV structural genes for construction of recombinant baculoviruses and VLP production in insect cells, well-known for allowing high yields of recombinant protein expression.

A potential strategy to produce safer and broadly protective influenza vaccines is to co-express, in the same cell host, multiple hemagglutinins (HA) with a matrix protein (M1) which self-assemble in virus-like particles (VLPs). This study demonstrates the suitability of combining stable expression and the baculovirus-expression vector system (BEVs) in insect Hi5 cells for production of such multi-HA Influenza VLPs. Stable pools of Hi5 cells expressing two HAs were generated and later infected with a M1-encoding baculovirus at two cell concentrations (CCIs; 2×106 cells/mL and 3×106 cells/mL). The HA concentration in culture supernatant was followed over time, with more productive infections observed at higher CCIs. To extend the culture time, a re-feed strategy was implemented based on the identification of key nutrients which were exhausted during cell growth. Afterwards, supplemented cultures infected at a CCI of 4×106 cells/mL allowed a 4-fold increase in HA concentration, at harvest, when compared to cultures infected at a CCI of 2×106 cells/mL. The production of multi-HA influenza VLPs using the aforementioned strategy could be successfully scaled-up to 2L bioreactor cultures with even higher volumetric (1.5-fold) HA yields. To surpass the unpredictability of gene expression promoted by the random integration strategy mentioned above, the recombinase-mediated cassette exchange (RMCE) technology was explored. The feasibility of having two cassettes flanked by distinct pairs of flippase recognition target sites (FRTs) was evaluated. Unfortunately, significant cross-interaction was observed between the selected pairs. To circumvent this bottleneck, a backup strategy consisting in the co-expression of two genes from the same locus after implementation of one cassette system, in insect Sf9 cells, was attempted. However, the isolated clones showed low expression of both M1 and HA proteins. Ongoing work focuses on the isolation of clones tagged in high expression loci by fluorescence activated cell sorter technology. This work demonstrates how the versatility of insect cell expression technology can be explored to produce Influenza VLPs as vaccine candidates.
A co-expressão de várias hemaglutininas (HA) e proteína da matriz (M1), no mesmo hospedeiro, formando partículas semelhantes a vírus (VLPs), constitui uma importante estratégia para desenvolver vacinas contra o vírus da gripe. Este trabalho mostra a combinação de uma linha celular estável de células de insecto com o sistema de expressão mediada por baculovírus para a produção deste tipo de VLPs. Foram estabelecidas duas populações de células de insecto Hi5, expressando duas HAs, posteriormente infectadas com um baculovírus contendo a proteína M1, a duas concentrações celulares diferentes (CCI; 2 e 3×106 cells/mL) sendo que a mais elevada demostrou ser a mais produtiva. De seguida, implementou-se uma estratégia baseada na adição de nutrientes específicos para prolongar o tempo de cultura. As culturas previamente suplementadas e infectadas a uma CCI de 4×106 células/mL produziram 4x mais HA comparativamente às culturas infectadas a uma CCI de 2×106 células/mL, não suplementadas. Esta estratégia foi também aplicada num biorreactor de 2L permitindo 1,5x mais produção, volumétrica, de HA comparativamente a experiências em pequena escala. De forma a ultrapassar a imprevisibilidade de uma integração aleatória, foi explorado o sistema de troca de cassete mediado por recombinase (RMCE). A viabilidade de um sistema com duas cassetes integradas flanqueadas por diferentes locais de reconhecimento (FRTs) foi avaliada, tendo sido observada a interação entre ambos os pares selecionados. Como segunda estratégia, foi implementado um sistema com uma cassete para co-expressão de dois genes em simultâneo, em células de insecto Sf9. Porém, os clones isolados mostram fraca expressão de M1 e HA, pelo que uma estratégia de isolamento de clones com expressão génica mais forte está em desenvolvimento utilizando uma tecnologia de sorteamento. Assim, este trabalho demonstra a versatilidade da tecnologia aplicada em células de insecto, que pode ser explorada para produzir VLPs multivalentes, com potencial para se tornar a próxima geração de vacinas para o vírus da gripe.

The Tetraviridae are a family of ss (+) RNA viruses that specifically infect lepidopteran insects. Their icosahedral capsids are non-enveloped and approximately 40 nm in diameter with T=4 quasi-equivalent symmetry. The omegatetraviruses, which are structurally the best characterised in the family, include Helicoverpa armigera stunt virus (HaSV) and Nudaurelia capensis omega virus (NwV). The omegatetravirus procapsid is composed of 240 identical copies of the capsid precursor proteins, which undergo autoproteolytic cleavage at its carboxyl-terminus generating the mature capsid protein (b) and γ-peptide. This process occurs in vitro following a shift from pH 7.6 to pH 6.0. The viral capsid encapsidates two ss genomic RNAs: The larger RNA1 encodes the viral replicase as well as three small ORFs while RNA2 encodes the capsid precursor protein together with an overlapping ORF designated P17. While a wealth of structural data pertaining to the assembly and maturation of omegatetraviruses is available, little is known about how this relates to their lifecycle. The principle aim of the research described in this thesis was to use an experimental system developed in the yeast, Saccharomyces cerevisiae, to investigate the assembly of HaSV and NwV virus-like particles (VLPs) in terms of maturation and encapsidation of viral RNAs, in vivo. The yeast expression system used two promoter systems for expression of capsid precursor protein: in the first, a hybrid promoter (PGADH) was used for high-level expression, while the second, PGAL1, produced substantially lower levels of the virus capsid protein precursors. An increase in the level of HaSV capsid protein precursor (p71) via the PGADH promoter resulted in a dramatic increase in VLP assembly as compared with the PGAL system. A protein equivalent to the mature capsid protein (p64) appeared at later time intervals following induction of transcription. Transmission electron microscopic studies showed that p64 correlated with the presence of mature VLPs as opposed to procapsids in cells containing p71. This confirmed that the presence of p64 denoted maturation of VLPs in vivo. Further investigation indicated that maturation correlated with cell aging and the onset of apoptosis. It was shown that induction of apoptosis resulted in VLP maturation while inhibition of apoptosis prevented maturation. These results suggested that the process of apoptosis might be the trigger for maturation of virus procapsids in their host cells. The increase in the efficiency of VLP assembly observed in the high-level expression system was proposed to be due to an increase in the cellular concentrations of viral RNA. To test this hypothesis, HaSV P71 was co-expressed with either P71 mRNA or full length RNA2. An increase in the solubility of p71 was observed in cells expressing increased levels of both RNAs, but there was no increase in the efficiency of VLP assembly. Northern analysis of encapsidated RNAs revealed that there was no selective encapsidation of either P71 mRNA or viral RNA2. This data indicated that the increase in viral RNA was not the reason for increased efficiency of VLP assembly, but most likely resulted from higher concentrations of p71 itself. It was decided to determine whether a highly efficient nodavirus replication system developed in yeast for heterologous production of proteins, could be used as a method for expressing the capsid protein precursor. The aim of using this system was to determine if VLPs assembled in a replication system specifically encapsidated viral RNA. Transcripts encoding the NwV capsid protein precursor (p70) were generated in yeast cells by replication of a hybrid RNA template by the Nodamura virus (NoV) replicase. Western analysis confirmed the presence of p70 as well as a protein of 62 kDa corresponding to the mature NwV capsid protein. Northern analysis of purified VLPs showed that NoV RNA1 and RNA3 were encapsidated, but no RNA2 was detected. Taken together, the data lead to the conclusion that specific encapsidation of tetraviral RNAs required more than close proximity of the viral RNAs and assembling virus-like particles. Encapsidation specificity in the omegatetraviruses may require additional viral proteins such as p17 during encapsidation or specific viral RNA encapsidation was replication-dependent. Replication-dependent assembly has been shown in the nodaviruses.

Problems associated with poor pharmacokinetics and biodistribution, as well as toxic off-target effects, limit the curative potential of most anti-cancer drugs. This has prompted the development of nanoparticulate drug delivery systems to impart both more favourable pharmacological properties and precise tumour targeting. The vast number of formulations, ranging from fully synthetic delivery systems to ones derived from natural sources, currently undergoing clinical trials or preclinical testing underlines the significance of this field. This project is a proof-of-concept investigation into the feasibility and effectiveness of a novel drug delivery system, based on virus-like particles (VLPs) of the MS2 bacteriophage. Doxorubicin (Dox) and an anti-BCL2 siRNA were used as model drug cargos. They were packaged inside MS2 VLPs either by chemical infusion, or via covalent attachment to an MS2 packaging signal, TR, respectively. An average loading of ~10 molecules of siRNA or ~110 molecules of Dox per VLP was achieved. Packaged cargos remained stably encapsidated; the siRNA was protected from nuclease degradation. VLPs were surface decorated with polyethylene glycol (PEG), and tumour-targeting ligands, human transferrin (Tf) or A9L, an RNA aptamer that targets prostate-specific membrane antigen (PSMA). Extensive PEGylation was achieved (~97% of coat proteins), and each VLP displayed on average ~7 molecules of Tf or ~16 molecules of A9L. PEGylation significantly reduced the non-specific cellular uptake of VLPs, and antibody binding. Further addition of tumour-targeting ligands facilitated the specific delivery of drug cargos to targeted cancer cells in culture, likely via receptor-mediated endocytosis, and induced significant cytotoxicity with an LC50 of ~10 nM for siRNA and ~800 nM for Dox. Importantly, negligible toxic effects were observed in the presence of excess free targeting ligands, or with non-targeted control cell lines. Furthermore, the cellular uptake of VLPs did not appear to induce any off-target effects. MS2 VLPs continue to show promise as a robust, flexible and effective drug delivery system. This project highlights the versatility of VLPs for displaying a range of useful ligands on their surface, as well as packaging various therapeutic cargos, and demonstrated their ability to specifically deliver drugs to targeted cancer cells. Though further studies are required, the work presented here is an important step towards fully realising the potential of this drug delivery system.

Molecular imaging involves the development of probes which can specifically label a certain object in the body at cellular or subcellular level. This thesis consists of three parts, each involving the development of novel labelling methods for viruses or virus-like particles with specific applications. Virus-like particles (VLP) derived from the E. coli bacteriophage Qβ are widely employed as a nano-carrier for drugs and vaccines, but a powerful method for tracing its circulation without affecting its structure is yet to be developed. In the first part of the thesis, the electrophilic fluorine source ¹⁹F-Selectfluor^TM was employed for introducing single fluorine atoms on Qβ VLPs. For the 'tag-and-modify' approach, site-selective electrophilic C-F bond formation was achieved on the dehydroalanine (Dha) amino acid tag of VLPs under aqueous conditions. Chemoselective electrophilic aromatic fluorination on tyrosine residues were also achieved using the same reagent by manipulating the amino acid sequence. Similar results were observed in conditions required for ¹⁸F-Selectfluor™ reaction, indicating the potential of this technique for positron emission tomography (PET) imaging. In addition, there is a lack of in situ technique for tracking the functional status of Qβ VLPs and hence the release of cargos. In the second part of the thesis, a simple way to monitor the disassembly of ¹⁹F-labelled Qβ VLPs by ¹⁹F NMR spectrosocpy is reported. Analysis of resonances, using experiments under a range of conditions, allowed determination not only of the intact particle but also the disassembled multimeric species and even smaller peptides upon digestion by cells. This in turn allowed mutational redesign of disassembly and testing in both bacterial and mammalian systems as a strategy for the creation of putative, targeted-VLP delivery systems. In the third part of the thesis, a new type of rhodamine B fluorescent dye functionalised with a 2-imino-2-methoxyethyl (IME) group is reported. The amidine linkage formed between the IME group and lysine residue retains the pKaH of the original side chain, which cannot be achieved using commercially available conjugating dyes. This in turn minimises the change in net charge hence virus infectivity following virus labelling. By employing adenovirus (AV) as an example, the IME dye was shown to be a better choice in retaining virus infectivity compared to dyes linked with other coupling groups. In addition, preliminary experiments on dengue virus with the synthesised dyes were also performed.

Includes abstract.
Includes bibliographical references (leaves 136-158).
HIV-1 Gag virus-like-particles (VLPs) produced in various expression systems are potent stimulators of both cellular and humoral immune responses in animal models. The encapsidation of large concentrations of random cellular RNA species is known to accompany the assembly of HIV virus particles. This RNA plays a crucial role by serving as a molecular scaffold for the assembly of Gag structural proteins into particles. Non-pseudotyped VLPs that do not present any HIV envelope glycoproteins are regarded as inert particles as they contain no replicative nucleic acid and are presumed to be unable to deliver encapsidated RNA for expression in inoculated individuals. Live virus cellular entry studies have shown that non-pseudotyped Gag particles are destined for degradation in acidified vesicles subsequent to receptor independent cellular entry. In addition to host cell RNA incorporation, Gag VLPs produced in insect cell-based, baculovirus expression systems have been observed to incorporate the baculovirus-derived Gp64 envelope glycoprotein. Gp64 has been shown to be efficient at enabling the delivery and expression of genes from recombinant baculoviruses and other Gp64 pseudotyped live viruses in mammalian cell lines both in vivo and in vitro. This study, therefore, set out to establish for the first time whether inert, baculovirus-derived (Gp64 pseudotyped) Gag VLPs could mediate delivery and expression of randomly encapsidated RNAs in mammalian cell lines.

Epizootic Hemorrhagic Disease Virus (EHDV) is an insect-transmitted pathogen of ruminants, causing periodic and significant losses in wild and captive deer populations and less frequently, a bluetongue-like disease in cattle. The serogroup of EHDV within the Orbivirus genus of the Reoviridae family consists of seven serotypes, in which emerging serotypes pose an increasing risk either regionally or globally, due to the insect vectors. To date, no vaccine against EHDV is commercially available, apart from the live-attenuated vaccine for EHDV-2 (IBAV). In this study, Virus-Like Particles (VLPs) of EHDV-1 and heterologous VLPs of EHDV-2 were generated using baculovirus multigene expression system for the synthesis of the two outer and two inner capsid proteins, essential for the formation of VLPs. The assembly of EHDV-1 recombinant structural proteins into Core-Like particles (CLPs, two proteins) and (VLPs, four proteins) was confirmed by EM analysis. The biological activity of the raised antisera to neutralise EHDV-1 was efficiently confirmed by neutralisation assay at 1:64 dilution. Cross neutralising activities were also detected against EHDV-2 and EHDV-6 serotypes at 1:8 dilution. Results presented in this study validate the potential efficacy of the VLP as a neutralising vaccine and strongly suggest its use as vaccine candidate. Additionally, an alternative approach was also initiated in this research to develop a rational vaccine against EHDV-2 using the reverse genetics system (RG). Towards this, it was first established that in vitro synthesised transcripts from purified EHDV-2 cores could generate infectious virus upon cell transfection. Note that both the generation of core transcripts and recovery of infectious virus of EHDV were not demonstrated previously. Subsequently a complete set of 10 T7 transcripts was synthesised, however, it was not possible to recover any infectious virus, likely to be some unwarranted mutations. Nevertheless, these transcripts will be further investigated for future RG studies.

Includes bibliographical references (leaves 114-128).
In this study I explored the possibility of making HIV-1 subtype C Pr55gag-based chimaeric virus-like particles (VLPs) as a boost to the HIV-IC multigene DNA vaccine pTHr.grttnC, which encodes a modified Gag-RT-Tat-Nef fusion protein (GRTTN). Furthermore, an attempt was made to produce VLP analogues to the HIV-IA polyepitope DNA vaccine pTHr.HIVA. A range of in-frame fusions with the C-termini of myristylation-competent p6-truncated Gag and native Pr55gag were made to test how the length of polypeptide and its sequence might affect VLP formation and structure.

Melanoma is the least common type of skin-cancer but the deadliest one. Cancer immunotherapy is considered a powerful tool in cancer treatment. Nevertheless, therapeutic cancer vaccines have led so far to modest immune responses with little clinical impact. The first part of this work is focused on developing a personalized melanoma vaccine platform based on VLPs and copper-free click chemistry, enabling bedside production of personalized cancer vaccines. There is currently a debate in the field whether cancer-specific, non-mutated germ-line epitopes or mutated neo-epitopes are more powerful antigens for vaccination. To address this issue, we identified melanoma-specific germ-line T-cell epitopes by immunopeptidomic and neoantigens using exome sequencing. Different VLP-based vaccine cocktails were generated, and their immunogenecity was compared in melanoma murine models. We demonstrate that both neoantigens and germ-line epitopes can induce partial protection, but the best therapeutic effect is achieved when combining both. The second part of the thesis tested a new adjuvant microcrystalline tyrosine (MCT) in combination with VLP-based vaccine (CuMV_TT). MCT, is an adjuvant that has been used in licensed allergen-immunotherapy products for decades. The adjuvant potential of MCT was also compared to Alum and B-type CpGs. MCT was similarly potent as CpGs and clearly superior to Alum when CD8⁺ T-cell responses and tumour protection were assessed. Combining a micron-sized adjuvant (MC) with nanoparticles (CuMVTT) appears a particularly attractive strategy as it optimally harnesses the drainage properties of the immune system. Thus, MCT may not only be used in marketed allergy products but may also be promising for induction of protective CTL response. Finally, we focused on delivering antigens and adjuvants in separate virus-like particles. This new technique would be beneficial when designing a personalized VLP-based vaccine as it would be handy to have a ready adjuvanted VLP which can be formulated with the patient vaccine at bed-side.

A thesis by Marc Ashley Bayliss entitled ‘Virus-like particles as a novel platform for delivery of protective Burkholderia antigens’ and submitted to the University of Exeter for the degree of Doctor of Philosophy. There is currently no licensed vaccine available for the global tropical pathogen Burkholderia pseudomallei which is the causative agent of melioidosis and a potential bio-threat agent. The capsule polysaccharide (CPS) expressed by B. pseudomallei has been shown to offer some protection against bacterial challenge. Polysaccharide immunogenicity can be enhanced by conjugation to a carrier protein and several licensed vaccines utilise this technology. Virus-like particles (VLPs) are non-infectious, non-replicating, viral proteins that self-assemble into viral structures and are in several licensed vaccines as primary antigens. VLPs are also effective delivery platforms for foreign antigens by genetic insertion or chemical conjugation. iQur, a collaborator on this project, has developed Tandem CoreTM that consists of two genetically linked hepatitis B core proteins that allow insertion of large proteins into each core whilst remaining assembly competent. The aim of this thesis was to assess the protective efficacy of Tandem CoreTM VLPs chemically conjugated to CPS and Tandem CoreTM Burkholderia protein fusion constructs. This involved three objectives; reduce the cost of CPS extraction; identify immunogenic Burkholderia proteins; and test candidate vaccine efficacy in an animal model of acute melioidosis against B. pseudomallei challenge. To reduce the cost of extraction, CPS was purified from B. thailandensis strain E555 and bacterial culture CPS concentration optimised which first required development of a quantitative ELISA. Immunogenic Burkholderia proteins were identified from the literature but Tandem CoreTM fusion constructs containing these proteins were not assembly competent. The Burkholderia proteins were added as co-antigens to the VLP CPS conjugate vaccine but did not improve efficacy. Tandem CoreTM VLPs conjugated to CPS were protective against B. pseudomallei challenge and were compared to CPS conjugated to Crm197: a commercially available carrier protein used in several licensed vaccines. At lower challenge doses, survival was greater in mice vaccinated with the VLP-CPS conjugate although at higher doses, Crm197-CPS efficacy was greater.

The final step of paramyxovirus infection requires the assembly of viral structural components at the plasma membrane of infected cells followed by budding of virions. While the matrix (M) protein of some paramyxoviruses has been suggested to play a central role in the assembly and release of virus particles, the specific viral and host protein requirements are still unclear. Using Newcastle disease virus (NDV) as a prototype paramyxovirus, we explored the role of each of the NDV structural proteins in virion assembly and release. For these studies, we established a virus-like particle (VLP) system for NDV. The key viral proteins required for particle formation and the specific viral protein-protein interactions required for assembly and release of particles were explored in chapter 2. First we found that co-expression of all four proteins resulted in the release of VLPs with densities and efficiencies of release (1.18 to 1.16 g/cm3and 83.8%±1.1, respectively) similar to that of authentic virions. Expression of M protein alone, but not NP, F-K115Q or HN proteins individually, resulted in efficient VLP release. No combination of proteins in the absence of M protein resulted in particle release. Expression of any combination of proteins that included M protein yielded VLPs, although with different densities and efficiencies of release. To address the roles of NP, F and HN proteins in VLP assembly, the interactions of proteins in VLPs formed with different combinations of viral proteins were characterized by co-immunoprecipitation. The co-localization of M protein with cell surface F and HN proteins in cells expressing all combinations of viral proteins was characterized. Taken together, the results show that M protein is necessary and sufficient for NDV budding. Furthermore, they suggest that M protein – HN protein and M protein - NP interactions are responsible for incorporation of HN protein and NP proteins into VLPs and that F protein is incorporated indirectly due to interactions with NP and HN protein. Since the vacuolar protein sorting (VPS) system is involved in the release of several enveloped RNA viruses, chapter 3 describes studies which explored the role of the VPS system on NDV particle release. First, we characterized the effects of three dominant negative mutant proteins of the VPS pathway on particle release. Expression of dominant negative mutants of CHMP3, Vps4 and AIP1 proteins inhibited M protein particle release as well as release of complete VLPs. Mutation of a YANL sequence in the NDV M protein to AANA inhibited particle release while replacement of this sequence with either of the classical late domain motifs, PTAP or YPDL, completely restored particle release. The host protein AIP1, which binds YXXL late domain sequences, is incorporated into M protein particles. These results suggest that an intact VPS pathway is necessary for NDV VLP release and that the YANL sequence is an NDV M protein L domain. The sequence and structure of the Newcastle disease virus (NDV) fusion (F) protein are consistent with its classification as a type 1 glycoprotein. We have previously reported, however, that F protein can be detected in at least two topological forms with respect to membranes in both a cell-free protein synthesizing system containing membranes as well as infected COS-7 cells (J. Virol. 2004 77:1951). One form is the classical type 1 glycoprotein while the other is a polytopic form in which approximately 200 amino acids of the amino terminal end as well as the cytoplasmic domain (CT) are translocated across membranes. Furthermore, we detected CT sequences on surfaces of F protein expressing cells and antibodies specific for these sequences inhibited red blood cell fusion to HN and F protein expressing cells suggesting a role for surface expressed CT sequences in cell-cell fusion. In chapter 4, we extended these findings and found that the alternate form of the F protein can also be detected in infected and transfected avian cells, the natural host cells of NDV. Furthermore, the alternate form of F protein was also found in virions released from both infected COS-7 cells and avian cells by Western analysis. Mass spectrometry confirmed its presence in virions released from avian cells. Two different polyclonal antibodies raised against sequences of the CT domain of the F protein slowed plaque formation in both avian and COS-7 cells. Antibody specific for the CT domain also inhibited single cycle infections as detected by immunofluorescence of viral proteins in infected cells. The potential roles of this alternate form of the NDV F protein in infection are discussed. Virus-like particles (VLPs) generated from different viruses have been shown to have potential as good vaccines. Chapter 5 explored the potential of NDV VLPs as a vaccine for NDV or as a vaccine vector for human pathogens. Significant quantities of NDV VLPs can be produced from tissue culture cells. These VLPs are as pure as virions prepared in eggs. In addition, some rules for incorporation of viral proteins into VLPs were also explored. We found that the cytoplasmic domain of the fusion (F) protein is necessary for its incorporation into VLPs. We found that an HN protein with an HA tag at its carboxyl terminus was incorporated into VLPs. We also found that the HN and F proteins of NDV, strain B1, can be incorporated into VLPs with M and NP of strain AV. The demonstration of specific domains required for protein incorporation into particles is important in using NDV VLPs as a vaccine vector for important human pathogens. In conclusion, this dissertation presents results that show that the M protein plays a central role in NDV assembly and release, a finding that is consistent with findings with other paramyxoviruses. More importantly, this work extends the current knowledge of paramyxovirus assembly and release by providing the first direct evidence of interactions between paramyxovirus proteins. These interactions between viral proteins provide a rational basis for incorporation of viral proteins into particles. This work also provides a clearer understanding of the role of the host vacuolar protein sorting machinery in NDV budding. A clear understanding of virus assembly and budding process contributes to the design of strategies for therapeutic intervention and in the development of safer, more economical and effective vaccines.

Las nuevas vacunas de subunidades están abriéndose paso dentro de la vacunología veterinaria y entre ellas, las pseudopartículas virales o VLPs (por su nombre en inglés “virus-like particles”) son una de las estrategias más atractivas que están abriendo nuevas fronteras en la vacunación de animales. Las VLPs son estructuras proteicas rígidas con un tamaño dentro del rango de los nanómetros, que presentan una geometría muy bien definida y una espectacular uniformidad que mimetiza la estructura de los virus nativos de los que proceden. Las VLPs tienen importantes ventajas en relación a la seguridad por el hecho de carecer de genoma viral, que elimina cualquier riesgo asociado a la replicación viral, reversión, recombinación o reorganizamiento genómico. La proteína de la cápside del virus de la fiebre hemorrágica del conejo (por su nombre en inglés “Rabbit haemorrhagic disease virus” o RHDV) es capaz de formar RHDVVLPs y estas partículas han demostrado poseer una fuerte inmunogenicidad, protegiendo al hospedador natural tras un desafío mortal. Además, estudios anteriores apuntaron la posibilidad de usar RHDV-VLPs como vector para mejorar la inmunoterapia contra el cáncer. Sin embargo, no hay estudios que hayan investigado la posibilidad de usar las RHDV-VLPs como vector vacunal con epitopos de enfermedades virales de animales. El objetivo de esta tesis es estudiar el potencial inmunogénico de las RHDV-VLPs como vectores vacunales de enfermedades virales en diferentes animales. En los dos primeros estudios, se investigó la inmunogenicidad de las RHDV-VLPs en el modelo murino, tanto in vitro como in vivo. Los resultados de estos estudios demostraron que el epítopo insertado era procesado y presentado mediante MHC-I por células dendríticas y que la inmunogenicidad dependía de los diferentes sitios de inserción. Las RHDV-VLPs quiméricas fueron capaces de proteger a los ratones frente a un desafío viral. También, la respuesta se vio alterada según la ruta de administración del antígeno. El tercer estudio confirmó los resultados en ratón, pero esta vez en experimentos in vitro con células de cerdo. Por último, se estudió inmunogenicidad de las RHDV-VLPs quiméricas en cerdo. Los resultados mostraron que la ruta de administración y el adjuvante determinaron la respuesta inmune después de la inmunización con las RHDVVLPs quiméricas y que los animales presentaban muy buena respuesta inmune celular y humoral, no solo frente a RHDV-VLPs sino frente al epítopo antigénico. Estudios posteriores se tendrán que abordar para demostrar la protección de los cerdos. En conclusión, en esta tesis se demuestra el potencial de las RHDV-VLPs como inmunógenos en dos sistemas animales diferentes.
New subunit vaccines are getting a foothold in veterinary vaccinology and virus-like particles (VLPs) are one of the most appealing approaches opening up frontiers in animal vaccines. VLPs are robust protein cages in the nanometer range exhibiting welldefined geometry and remarkable uniformity that mimic the overall structure of the native virions. VLPs have an important advantage in terms of safety; indeed, lacking the genome of the virus avoid any of the risks associated with virus replication, reversion, recombination or re-assortment. Rabbit haemorrhagic disease virus (RHDV) capsid protein is able to form RHDV-VLPs and these particles showed a strong immunogenicity and protected the natural host after a lethal challenge. Additionally, previous studies described the possibility to use RHDV-VLPs as platform for the insertion of foreign epitopes or for DNA packaging. Nowadays, one study has shown the possibility to use RHDV-VLPs as carrier for improving cancer immunotherapies but no studies have investigated the possibility to use RHDV-VLPs as vaccine vectors carrying epitopes corresponding to viral animal diseases. This thesis is aimed to study the potential immunogenicity of RHDV-VLPs as epitope carriers for viral disease in different animal models. In the first two studies, the immunogenicity of chimeric RHDV-VLPs was investigated in a murine system in vitro and in vivo. Results from these studies demonstrated that the inserted epitope was processed and presented in an MHC-I context by dendritic cells (DCs) and that the different sites of insertion of the epitope influenced the immunogenicity of the VLPs. Chimeric RHDV-VLPs were able to protect mice from a viral challenge. Also, the route of antigen delivery influenced the immunogenicity of the particles. The third study confirmed the initial results but this time in in vitro experiments using porcine cells. Lastly, chimeric RHDV-VLPs were studied as immunogens in pigs. The results showed that the delivery route and adjuvant influenced immune responses after chimeric RHDV-VLP immunization and more importantly that pigs exhibited very good cellular and humoral immune responses against not only RHDV-VLPs but also against the antigenic epitope. Further studies have to be performed to prove protection in pigs. In conclusion, in this thesis we demonstrated the potential of RHDV-VLPs as immunogens in two different animal systems.

Les virus-like particles (VLPs) derivades de l’HIV presenten un gran potencial com a vacunes de nova generació. La proteïna Gag s’ensambla de forma espontània a la membrana cel·lular, donant lloc a VLPs amb embolcall. Per a generar VLPs en suficient quantitat i amb la qualitat requerida per a estudis clínics, es necessiten processos de producció robustos. A més, la disponibilitat de mètodes analítics senzills i confiables és crítica per al desenvolupament, optimització i monitorització d’aquests processos. Les cèl·lules de mamífer són la plataforma preferida per a produir VLPs, ja que proporcionen l’ambient necessari per a la seva correcta formació i ensamblatge. En aquest treball, es va desenvolupar un procés de producció per a VLPs de Gag-GFP mitjançant la línia cel·lular CAP, que destaca per la seva novetat. El primer pas va ser el desenvolupament d’una tècnica de quantificació basada en fluorescència, per a poder-la utilitzar de forma rutinària en el desenvolupament del bioprocés. A més, el mètode es va validar d’acord a l’ICH. El mètode era específic per a Gag-GFP i les mesures de fluorescència correlacionaven amb les mesures d’ELISA de referència. El mètode era adequat ja que mostrava poca variabilitat, un rang de treball de 3 ordres de magnitud i un límit de detecció suficientment petit com per a poder quantificar mostres sense purificar. La producció de VLPs es va dur a terme mitjançant transfecció transitòria amb PEI a línia cel·lular CAP-T. El procés a petita escalar es va optimitzar de forma sistemàtica. Primer, algunes variables es van estudiar per separat per a establir les condicions inicials. La falta d’un medi de cultiu compatible amb el creixement cel·lular i la transfecció forçava la realització d’un recanvi de medi abans de la transfecció. La densitat cel·lular en el moment de la transfecció, i les concentracions de DNA i de PEI es van optimitzar mitjançant una metodologia de Disseny d’Experiments. El títol aconseguit va ser de 6×1010 VLP/mL, amb les característiques desitjades. A continuació, es va dur a terme l’escalat del procés de transfecció a bioreactor d’1L, centrat en dos aspectes principals: el creixement cel·lular amb un estat fisiològic òptim i evitar el recanvi de medi per centrifugació abans de la transfecció, ja que no és desitjable a escales grans. Les cèl·lules CAP-T es van cultivar en bioreactor controlant el pH amb bicarbonat de sodi i amb aeració superficial. El recanvi de medi es va evitar cultivant les cèl·lules en una combinació de medis (FreeStyleF17 + 1% de PEM) compatible amb el creixement cel·lular i la transfecció transitòria. Aquesta innovadora estratègia va permetre simplificar significativament la transfecció transitòria a gran escala, mentre que es van aconseguir un bon creixement cel·lular, bona eficiència de transfecció i una producció de VLPs d’alta qualitat comparable al sistema a petita escala. El següent pas va ser la generació d’una línia cel·lular per a produir de forma estable les VLPs. La metodologia que es va utilitzar va ser la limiting dilution. Es van analitzar ~80 clons per a seleccionar el més productiu. A més, es va millorar la productivitat del clon seleccionat mitjançant sorting de les cèl·lules amb major intensitat de GFP. El cultiu d’aquest clon en bioreactor en fedbatch va donar lloc a un títol de 2×109 VLP/mL. Finalment, es va utilitzar l’espectroscòpia dielèctrica per a monitoritzar in-line els dos processos de producció de VLPs, basats en expressió estable i transient. La mesura de la permitivitat va permetre monitoritzar la densitat cel·lular en temps real. A més, els paràmetres de la corba de dispersió β mostraven perfils específics que es relacionaven amb events crítics durant el bioprocés, com ara canvis metabòlics o mort cel·lular.
HIV-1 virus-like particles (VLPs) have great potential as new generation vaccines. Upon expression, the Gag polyprotein of HIV-1 assembles spontaneously in the vicinity of the plasma membrane giving rise to enveloped VLPs. Robust production processes are required to generate VLPs of sufficient quality and quantity for pre-clinical and clinical evaluation. In addition, the availability of simple, fast and reliable analytical tools is critical to develop, optimize and monitor such processes. Mammalian cells are the preferred platform to produce VLPs, since they provide the required environment for their correct formation and assembly. In this work, a production process for Gag-GFP VLPs was developed using the novel CAP cell line. The first step was the development of a simple and cost-effective fluorescence-based quantitation technique that could be used routinely for bioprocess development. In addition, the method was validated according to ICH guidelines. The method showed to be specific for Gag-GFP and the fluorescence signal correlated well with the measurements of a reference ELISA assay. Little variability compared to ELISA, linearity over a 3-log range and a sufficiently low limit of detection to quantify crude samples demonstrated the suitability of the technique. The CAP-T cell line was used to produce Gag-GFP VLPs by PEI-mediated transient transfection. A systematic optimization of the process at small scale was performed. First, some variables were studied separately to set the conditions. The lack of a culture medium that was compatible with suitable cell growth and PEI-mediated transient transfection, forced the performance of a medium replacement step prior to transfection. Then, the cell density at the time of transfection, and the DNA and PEI concentrations were optimized by means of a Design of Experiments approach. The final titer obtained was 6×1010 VLP/mL, with the desired quality attributes. The scale-up of the transient transfection protocol to 1L bioreactor followed, focusing in two main aspects: cell culture with optimal cell physiological conditions and avoiding the medium replacement step by centrifugation prior to transfection, which is not desirable at large-scales. CAP-T cells were cultured in bioreactor by addition of carbonate to control pH and surface aeration. Remarkably, the medium replacement step was avoided by culturing the cells in a combination of media (FreeStyleF17 + 1% of PEM) compatible with both cell growth and PEImediated transient transfection. This novel strategy significantly simplified large-scale transient transfection, while suitable cell growth, transfection efficiency and high quality VLP production comparable to small scale were achieved. The generation of a stable CAP cell line producing correctly assembled Gag-GFP VLPs was the next step. The methodology employed was the classic limiting dilution, which has the advantages of being straightforward and easily implementable at any cell culture lab. A screening of ~80 clones was performed to select the most productive one. Moreover, the specific productivity of the selected clone was further improved by sorting the most intense GFP expressing cells. The culture of this clone at bioreactor level in fed-batch mode was successful, obtaining a VLP titer of around 2×109 VLP/mL. Finally, dielectric spectroscopy was employed for the in-line monitoring of the two VLP production processes set in this work, based on transient and stable expression. The measurement of the permittivity during the culture allowed to monitor the cell density in real time. Moreover, specific profiles of the β-dispersion parameters were identified, and related to critical events in the bioprocess, such as metabolic shifts or cell death.

The objective of this study was to understand the mechanism of lipid fouling in chromatography through the investigation of a hydrophobic interaction chromatography (HIC) operation. This was motivated by the need to understand this phenomenon during the manufacture of biological products such as vaccines. The systematic approach and novel analytical techniques employed create a unique platform to study fouling of other chromatographic adsorbents and process feed materials. HIC is employed as a primary capture step in the purification of yeast derived hepatitis R surface antigen (HBsAg), where the required cell disruption and detergent liberation steps release high levels of lipid content into the feed stream. From lipid- rich and lipid-depleted feedstocks, comparative analysis was able to quantify the deterioration in HIC performance (binding capacities, purities and recoveries) under successive cycles. Furthermore, a full mass balance on host lipids identified the highly hydrophobic triacylglyceride as the main foulant. Intra-particle distribution and progression of lipid fouling and its effects on material adsorption and diffusion were then examined under confocal laser scanning microscopy (CLSM). In addition, high- resolution scanning electron microscopy (SEM) images of the fouled bead (after 40 cycles) confirmed that a thick lipid layer was building up on the outer bead surface. Based on these findings, the mechanism of fouling was thought to be the rapid accumulation of lipid foulant at the rim of the bead, which was aggravated by the possible diffusion hindrance resulting from multi layer adsorption. Finally, pretreatments to reduce this mechanism of chromatography fouling were evaluated in terms of improvement on feed quality and HIC performance. Selective adsorbent polystyrene XAD-4 demonstrated promising lipid removal capabilities with satisfactory HBsAg VLP recoveries. The improved feed into the column resulted in a three-fold increase in product capacity, whilst the overall yield remained constant over 40 cycles.

Cervical cancer is caused by infection with high-risk Human papillomaviruses (HPVs). It is ranked fourth among the top cancers in women worldwide, with ~87% of the global cervical cancer cases reported in developing countries. The HPV L1 capsid protein can self-assemble into virus-like particles (VLPs) that are structurally like native virions, which is the foundation on which commercially available vaccines have been developed. There are 3 commercially available HPV vaccines that are effective at preventing HPV infections, but are expensive, therefore limiting their use in the poorer developing countries where they are most needed. Thus, there is a need for more cost-effective HPV vaccines for use in these countries. Over the years, the use of plants to produce vaccines has begun to be more favourably looked upon as a costeffective alternative to conventionally used expression systems. The aim of this study was to evaluate the plant-based transient expression system as a tool to produce potentially cost-effective HPV L1 VLP-based vaccines, particularly for developing countries. Firstly, the L1 proteins of the 8 most common high-risk Human papillomavirus types in Africa (HPV 16, 18, 31, 33, 35, 45, 52, and 58) and 2 low risk types (HPV 6 and 34) were transiently expressed in Nicotiana benthamiana. The proteins were purified via isopycnic ultracentrifugation using sucrose and Optiprep™ density gradients, and the assembly of VLPs assessed by transmission electron microscopy (TEM). To further assess whether the VLPs are immunogenic, HPV 35, 52 and 58 were selected for mice studies. These were selected in particular, as HPV 35 is the fifth most prevalent type in Africa, and HPV 52 and 58 are among the most frequently reported high-risk types in Sub-Saharan Africa. VLPs representing the 3 HPV types were quantified and prepared for immunization in mice. The commercially available Gardasil® HPV VLP vaccine was used as a positive control. The immunogenicity of the vaccines was evaluated by testing for the presence of anti-L1 antibodies in sera from immunized mice using enzyme-linked immunosorbent assays (ELISAs) and western blots. Sera from immunized mice were also tested for the presence of neutralizing antibodies using pseudovirion based neutralization assays (PBNAs). L1 proteins of all 10 HPV types tested were successfully expressed in N. bethamiana, and TEM analysis showed that expression resulted in the successful formation of fully assembled VLPs (40-60nm) as well as small VLPs and/or capsomeres (25-39nm). The analysis of the immune response showed that type-specific L1-specific antibodies were produced which were able to successfully neutralize homologous pseudovirions (PsVs) in PBNAs. Sera from mice immunized with plant-produced VLPs were further tested against heterologous HPV 6, 16, 18, 31, and 45 PsVs. However, none of the tested heterologous HPVs were neutralized, suggesting that plant-made VLPs induced type-specific neutralizing antibodies only. In conclusion, this study successfully demonstrated the potential for using plant-based transient expression systems to produce affordable and immunogenic HPV vaccines, particularly for developing countries. This is the first study describing the expression of 10 HPV L1 proteins in plants, marking a step towards the development of cheaper HPV vaccines which could be combined to generate an effective multivalent vaccine against HPVs.

Current influenza vaccine manufacturing processes using chicken-embryonated egg technology is a time-consuming and laborious process, and is currently the major drawback in counteracting pandemic influenza strain. One solution to that problem is the use of plants to generate vaccine antigen. Virus-like particles (VLP), produced from the tobacco plant Nicotiana benthamiana, represent a cost-effective, alternative platform for influenza vaccine production. Previous studies have shown that the immunization with VLP expressing the hemagglutinin (HA) protein from influenza virus H5N1 (H5-VLP) produced in N. benthamianainduce protective immunity against challenge of cross-clade virus in mice and ferrets. In this study, we used human peripheral blood mononuclear cells (PBMC) to characterize the innate immune response to plant-derived influenza H5-VLP ex vivo. We successfully demonstrate the mitogenic property of H5-VLP on PBMC ex vivo. Furthermore, we detect up-regulation of activation marker in B cells and NK cells, and some T cells. Cytokine profile of the supernatant from VLP-stimulated sample suggests that inflammatory response dominates the innate immunity within first 48 hours and is produced by CD14+ monocytes. Our study demonstrates that tobacco plant-derived influenza VLP are capable of generating innate immune responses in naïve human PBMC, helping us to better understand the immunostimulatory nature of this potential vaccine candidate.
A l'heure actuelle, la plupart des vaccins contre les infections par le virus influenza sont produits à partir d'œufs de poule fécondés. Ce procédé long et fastidieux constitue l'un des principaux obstacles à la production rapide d'un vaccin lors d'une pandémie. Une solution à ce problème consiste en l'utilisation de plantes afin de générer les antigènes nécessaires à l'élaboration du vaccin. Les pseudovirus ou Virus-like particles (VLP) produites à partir de la plante de tabac Nicotiana benthamiana représentent une alternative moins couteuse et plus rapide pour la production de vaccins antigrippaux. Des études préalables ont démontré qu'une immunisation avec les VLP exprimant l'hémagglutinine (HA) du virus influenza H5N1 (H5-VLP) induisaient une immunité protective lors d'une infection par ce virus chez la souris et le furet. Dans notre étude, nous avons utilisé les cellules mononuclées du sang périphérique humain (PBMC) afin de préciser la réponse immunitaire innée suite à l'exposition ex vivo aux H5-VLP produites dans N. benthamiana. Nous avons démontré les propriétés mitogéniques des H5-VLP sur les PBMC ainsi qu'une activation des lymphocytes B, des cellules NK et de certaines sous populations de lymphocytes T. L'analyse des cytokines sécrétées dans le surnageant des PBMC exposés ex vivo aux VLP suggère qu'une réponse pro-inflammatoire prédomine 48h après exposition et semble résulter essentiellement d'une activation des monocytes CD14+. Notre étude démontre que les VLP produites à partir de la plante de tabac génèrent une réponse immunitaire innée dans les PBMC provenant de patients naïfs, nous permettant ainsi de mieux comprendre les propriétés immunostimulantes de ce nouveau type de vaccin.

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.

Cette thèse s'est développée selon deux axes de recherche: l'amélioration de la détection des norovirus (humains et animaux) et des rotavirus, et l'évaluation du potentiel des VLPs comme substitut viral pour l'étude de leur devenir en milieu marin. Le point majeur concernant l'optimisation de la détection est la mise au point de la détection des norovirus par RT-PCR en temps réel. La seconde partie du travail a permis de mettre en évidence le potentiel des VLPs de RV et de NV à se substituer aux virus natifs, prouvé par une étude de stabilité des particules en eau de mer naturelle. Grâce à leur caractère non pathogène, la longue persistance des VLPs RV dans les coquillages a été démontrée en conditions naturelles (huîtres sur estran). Les études de fixation des VLPs NV sur les tissus d'huîtres ont montré une fixation spécifique sur divers tissus
This work was developed following two research axes: the improvement of norovirus (human and animal strain) and rotavirus detection, and the evaluation of VLPs as a viral substitute for studying their comportment in marine environment. The main point concerning the method optimisation was the development of real-time RT-PCR for norovirus detection. The second part of the work has demonstrated the potential of norovirus and rotavirus VLPs as a viral surrogate as proven by the study of their stability in seawater. As VLPs are non-pathogen particles, the long persistence of rotavirus VLPs in shellfish has been shown in natural conditions (oysters on marine estuaries). The studies of Norwalk virus VLPs attachment to oyster tissues have demonstrated a specific binding to some tissues

Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Programa de Pós-Graduação em Biologia Molecular, 2016.
Texto liberado parcialmente pelo autor. Conteúdo restrito: Capítulo 2 e Anexos.
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Os baculovírus são vírus de insetos amplamente utilizados no controle biológico de pragas agrícolas e também como ferramenta para expressão de proteínas heterólogas. Os baixos custos, a segurança na manipulação e as diferentes formas de expressão de proteínas tornam os baculovírus e células de inseto uma escolha eficaz para a correta expressão de antígenos vacinais e produção de VLPs (“Virus like particles”). No capítulo 1 deste trabalho, foi avaliado o potencial imunogênico de BVs (“Budded virus”) recombinantes contendo o EDIII (Domínio III da proteína de envelope do vírus da Febre amarela – FA) fusionado à proteína GP64 dos baculovírus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) e Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV); e também de uma massa protéica gerada pela fusão de EDIII com a proteína poliedrina de AcMNPV. O EDIII interage com os receptores celulares e contém os epítopos reconhecidos pelos anticorpos neutralizantes, sendo assim alvo para a produção de uma vacina de subunidade. Foi mostrado neste trabalho, a confirmação da correta expressão das proteínas recombinantes (GP64 + EDIII e Poliedrina + EDIII) por Western blot, microscopia de luz e confocal. No caso do EDIII fusionado à GP64 de AcMNPV e AgMNPV, as partículas virais recombinantes foram purificadas e inoculadas em camundongos. O teste de proliferação de linfócitos indicou uma maior proliferação em camundongos inoculados com o vírus recombinante contendo o EDIII fusionado à proteína GP64 do baculovírus AgMNPV quando comparado com o controle LPS. Testes complementares serão feitos, para avaliar o perfil da resposta imunológica e confirmar a obtenção de um antígeno vacinal contra FA. No capítulo 2, o sistema baculovírus de expressão foi utilizado para expressar o precursor da proteína GAG (Pr55) de HIV-1. A expressão de Pr55 HIV-1 é suficiente para a montagem de VLPs na membrana plasmática da célula do hospedeiro. Porém, a proteína GP64 de baculovírus é altamente imunogênica e também é expressa na superfície de células infectadas e normalmente está presente na superfície da VLP. Neste trabalho, mostramos que não é possível separar BVs e VLPs produzidos em um mesmo ciclo de infecção por baculovírus mesmo usando diferentes metodologias de separação. Então, utilizando um sistema que consegue produzir baculovírus recombinantes livres de GP64, foi construído o vírus recombinante vGAGHIV-1 GP64 null e utilizado na infecção de células Sf9. Por Western blot foi observado a perda do sinal da proteína GP64 de baculovírus no extrato de células infectadas com esse vírus recombinante. Essas mesmas células foram visualizadas por microscopia eletrônica de transmissão (MET), mostrando que mesmo quando GP64 não está presente (o que resulta na ausência de produção de BVs), o brotamento de VLPs continua a acontecer. Essa técnica mostrou-se eficiente para a produção de VLPs livres de partículas ou proteínas baculovirais. Este trabalho confirma a eficiência do uso do sistema de expressão baseado em baculovírus para a expressão de proteínas e VLPs de interesse famacológico. _________________________________________________________________________________________________ ABSTRACT
Baculoviruses are insect viruses widely used in the biological control of agricultural pests and as a tool for heterologous protein expression. Their low production costs, security in manipulation and the many ways of protein expression, make baculoviruses and insect cells an effective choice for the efficient expression of vaccine antigens and VLPs (Virus like particles) production. In chapter 1 of this work, it was evaluated the immunogenic potential of recombinant BVs (Budded virus) containing EDIII (Yellow fever virus -YF- envelope protein domain III) fused to the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) GP64 protein; and also of a protein mass generated by the fusion of the EDIII and AcMNPV polyhedrin protein. The EDIII interacts with cell receptors and contains epitopes recognized by neutralizing antibodies, being the target for the production of a subunit vaccine. We showed in this work the confirmation of the correct recombinant protein expression (GP64 + EDIII and Polyhedrin + EDIII) by Western blot, optical and confocal microscopy. In the case of the EDIII fused with AcMNPV and AgMNPV GP64, the recombinant viral particles were purified and inoculated in mice. The lymphocyte proliferation assay showed a higher proliferation in mice immunized with recombinant virus containing the EDIII fused with the GP64 from the AgMNPV baculovirus comparing to the LPS control. Complementary assays will be carried out in order to evaluate the immunological response pattern and to confirm the production of a vaccine antigen against YF. In chapter 2, the baculovirus expression system was used to express the GAG protein precursor (Pr55) of HIV-1. The Pr55 expression is sufficient to VLP assembly on the cell host plasma membrane. However, the GP64 baculovirus protein is highly immunogenic and is also expressed on the surface of infected cells and is also present on the surface of the VLP. In this work, we showed that is not possible to separate BVs and VLPs produced in the same baculovirus infection cycle using different separation methodologies. Then, using a system able to produce GP64 free recombinant baculovirus, the recombinant virus vGAGHIV-1 GP64 null was produced and used to infect Sf9 cells. By Western blot analyses, it was shown that the baculovirus protein GP64 signal was missing in the recombinant virus infected cell extract. These same cells were observed by transmission electron microscopy (MET), showing that even when the GP64 is absent (which results in the absence of BVs production), VLPs budding continues to happen. This technique was shown to be efficient for VLPs production, free of baculvirus particles or proteins. This work confirms the efficiency of the baculovirus expression system for protein expression and VLPs of pharmacological interest.

Influenza viruses represent a significant public health problem, since they are the etiological agents of acute respiratory illness that can occur in annual epidemics or in occasionally pandemic events. Influenza viruses constantly evolve by the accumulation of point mutations at the level of their antigenic determinants hemagglutinin and neuraminidase (antigenic drift), or as a result of a genetic reassortment (antigenic shift), usually happening when avian and human viruses co-infect an intermediate host. The high rate of mutations, the possibility of reassortment of gene segments and the wide variety of influenza viruses explain their ability to overcome the host immunity, acquired by previously vaccination (Wong et al., 2005). Moreover, the difficulty of developing vaccines capable of inducing a long-lasting immune response leads to the need of up-dating the vaccine formulation every year (Wong et al., 2005). On the other hand, molecular evolution processes determine an increasing emergence of drug-resistant viruses that restrict the efficacy of the antiviral drugs. Indeed, vaccination remains the most effective preventative strategy to reduce the chance of infection from influenza virus and its complications. In this context, vaccines based on virus-like particles (VLPs) are extremely promising. In fact VLPs, while being antigenically identical to the infectious virus, lack pathogenicity. VLPs are obtained by expressing specific viral structural proteins, that display the ability to bud from cells, in the absence of any other viral component (such as retroviral Gag and papillomavirus L1) (Zeltins, 2013). VLPs can be used per se or as “platforms” for the presentation of immunodominant epitopes. In the case of influenza viruses, the matrix protein M1 represents the major driving force of viral budding process (Gomez-Puertas et al., 2000). However, the expression of M1 alone does not allow the production of VLPs. Among other reasons, this feature is due to the lack in M1 of a membrane targeting signal (Wang et al., 2010a). In this context, during the viral infection, it is the second matrix protein, M2, which is charachterized by a targeting signal and can physically interact with M1, that brings the latest in contact with the plasma membrane (Chen et al., 2008). Moreover, M2 is also able to facilitate the curvature of the membrane and the scission of the envelope of the budding viral particle from the cell surface (Rossman et al., 2010), a function that is linked to the cellular complex ESCRT (endosomal sorting complex required for transport)-III, in the case of most enveloped viruses (Calistri et al., 2009b). The overall aim of our work was to optimize the production of influenza VLPs based on M1, by identifying the minimal sequences requested to render M1 able to bud independently from M2 co-expression. To this end, we created different recombinant M1 proteins characterized by the ability to interact with different ESCRT components and to directly contact the plasma membrane, in the absence of M2. Specifically, we generated constructs expressing M1 fused to the myristilation signal of HIV-1 Gag and containing short proline rich domains (Ldomains) capable of interacting with either Tsg101 (PTAP), AIP1/Alix (YPDL), both cellular proteins able to interact with ESCRT-III and involved in the budding of several RNA enveloped viruses (Calistri et al., 2009b), or to a motif (PTAPPEY) known to be extremely efficient in mediating the production of VLPs (Strack et al., 2000). Moreover, we directly fused at the C-terminus of M1 specific ESCRT-III proteins, namely CHMP3 and CHMP4B. In this way, we were able to demonstrated that: i) the HIV-1 Gag myristilation signal, added to the N-terminus of M1 (Myr-M1), confers it the ability to contact the plasma membrane; ii) the insertion of the small proline rich domain PTAP allows Myr-M1 to form VLPs and iii) the fusion between M1 and the cellular protein CHMP4B allows M1 to bud from the cells, independently from the presence of the myristilation signal. Furthermore, we were able to demonstrated that M2 alone and the M2-M1 fusion protein can self-assemble and bud into virus-like particles. In this context, by engineering specific mutants of the Gag proteins of the feline immunodeficiency virus (FIV), previously described in our laboratory (Calistri et al., 2009a), we were able to demonstrated that indeed M2, and in particular its cytoplasmic region, can act as a substitute of ESCRT-III components for the budding of the influenza virus. Finally, VLPs based on M2, M2-M1 and M1-Myr-CHMP4B, were used in a pilot experiment performed in mice, in order to analyze their immunogenicity. Interestingly, we were able to detected specific antibodies directed against the protein M1, even though in a small number of tested animals. However, the obtained data are encouraging, since the purification of the employed VLPs, which are still under characterization in terms of density and morphology, was not optimal, thus affecting the obtained results. Finally, we also generated different hemagglutinin (HA) mutants characterized by the absence of the globular head (headless-HA). Indeed, it is well known that the conserved stalk domain of influenza HA is highly immunogenic and confers a broadly immune response (Steel et al., 2010). We demonstrated that headless HA can be incorporated into engineered VLPs, based on FIV Gag protein. Currently, we are analyzing the possibility of incorporating headless-HA protein also in M1-based VLPs. Next, after setting up the appropriate purification protocol, we would test these M1- based VLPs, together with the FIV-based one, in the mouse model. In conclusion, overall our data provide on one hand important biological insights into the function/characteristics of the influenza virus M1 and M2 proteins and, on the other hand useful information for the development and improvement of influenza-based VLPs to be employed in vaccination strategies.
I virus dell’influenza costituiscono un rilevante problema di sanità pubblica a livello mondiale, in quanto sono gli agenti eziologici di patologie respiratorie acute che si possono presentare sotto forma di epidemie annuali, oppure come pandemie occasionali. Questi virus evolvono costantemente mediante l’accumulo di mutazioni puntiformi a carico degli antigeni di superficie emoagglutinina (HA) e neuraminidasi (NA) (antigenic drift), oppure in seguito a fenomeni di riassortimento genico (antigenic shift) derivanti dalla coinfezione di virus aviari ed umani in un ospite intermedio. L’alto tasso di mutazioni, la possibilità di riassortimento dei segmenti genici e l’ampia varietà di virus dell’influenza spiegano la capacità di questi virus di superare le barriere anticorpali indotte da immunità pregresse (Wong et al., 2005). Inoltre, l’alta variabilità del virus dell’influenza limita anche l’efficacia della terapia antivirale a disposizione a causa dei frequenti fenomeni di farmaco-resistenza. La vaccinazione rappresenta, quindi, la miglior strategia per prevenire e ridurre le possibilità di infezione da parte del virus dell’influenza e le conseguenti complicazioni. Tuttavia, le strategie per lo sviluppo di vaccini efficaci, capaci di stimolare immunità di lunga durata, sono risultate vane ad oggi, determinando quindi la necessità di cambiare ogni anno la formulazione del vaccino influenzale (Wong et al., 2005). Per questo motivo, sono allo studio nuovi approcci vaccinali che consentano di ottenere, una volta inoculati nell’uomo, una protezione prolungata e diretta contro diversi sottotipi influenzali (vaccini universali). Inoltre, si cerca di migliorare l’efficacia e la rapidità delle procedure produttive dei vaccini classici, in maniera da poter affrontare, se necessario, emergenze quali l’insorgenza di ceppi influenzali pandemici (Kang et al., 2011). Una tecnica molto promettente, da entrambi i punti di vista, risulta essere quella basata sulla produzione di particelle simil-virali o Virus-like particles (VLP). Le VLP mimano strutturalmente la particella virale, ma mancano della capacità replicativa, poichè prive del genoma virale (Zeltins, 2013). Le particelle simil-virali possono essere ottenute mediante l’espressione delle proteine strutturali virali (es.: Gag dei retrovirus, L1 di HPV), le quali hanno la capacità intrinseca di autoassemblarsi e gemmare dalle cellule (Haynes, 2009). Inoltre, le VLP posso essere utilizzate anche come piattaforme per la presentazione di epitopi, anche eterologhi, immunodominanti. La proteina di matrice M1 rappresenta la driving force della fase di gemmazione del virus dell’influenza (Gomez-Puertas et al., 2000). La sua espressione nelle cellule, tuttavia, non è sufficiente a determinare la produzione di VLP. Questo dipende da diverse ragioni, tra le quali l’incapacità di M1 di contattare da sola la membrana plasmatica, a causa della mancanza di un segnale specifico di indirizzamento (Wang et al., 2010a). A questo proposito, è stato dimostrato come un’altra proteina strutturale virale, M2, svolga un ruolo importante nelle ultime fasi del processo di gemmazione virale, mediando proprio l’interazione tra M1 e la membrana plasmatica (Chen et al., 2008). Inoltre, è stato recentemente chiarito che la capacità del virus dell’influenza di gemmare dalle cellule infettate in assenza del pathway di biogenesi dei Multivescicular Bodies (MVB), funzionale, utilizzato invece dalla maggior parte dei virus ad RNA dotati di envelope, sia da ascrivere proprio a funzioni intrinseche di M2. In particolare, M2 sarebbe in grado di funzionare come sostituto delle proteine appartenenti al complesso ESCRT (endosomal sorting complex required for transport)-III, fondamentale nell’ambito della formazione dei MVB e nella gemmazione di molti virus a RNA (Rossman et al., 2010). Scopo del presente lavoro di tesi è stato quello di conferire ad M1 le minime caratteristiche necessarie per gemmare da cellule eucariotiche anche in assenza di altri componenti virali. A tale scopo, M1 è stata opportunamente ingegnerizzata attraverso la fusione di domini, porzioni o intere proteine eterologhe, al fine di conferirle la capacità di contattare autonomamente la membrana cellulare e di sfruttare ESCRT-III. Il presente lavoro di dottorato si propone quindi l’ottimizzazione della produzione di VLP del virus dell’influenza come piattaforma per lo sviluppo di un vaccino universale. In particolare, l’attenzione è stata focalizzata sulla proteina di matrice M1 e sulla possibilità di renderla indipendente dalla proteina M2 durante la fase di gemmazione. È stato possibile quindi dimostrare: i) che il segnale di miristilazione della proteina Gag del virus dell’immunodeficienza umana (HIV-1), fuso nella regione amminoterminale di M1 (Myr-M1), le conferisce la capacità di contattare la membrana plasmatica, ii) che l’aggiunta di un piccolo dominio ricco in prolina (PTAP), noto in letteratura come in grado di mediare l’interazione tra proteine diverse e il pathway di biogenesi dei MVB, consente ad Myr-M1 di gemmare nel sovratante cellulare; iii) che la fusione tra M1 e la proteina cellulare CHMP4B, appartenente al complesso ESCRT-III consente ad M1 di fuoriuscire dalle cellule, indipendentemente dalla presenza del segnale di miristilazione. Inoltre, abbiamo dimostrato che la proteina di fusione M2-M1 è in grado di gemmare autonomamente dalle cellule, così come la sola M2. Infine, le VLP basate su M2, M2-M1 e Myr-M1-CHMP4B sono state utilizzate in un esperimento pilota, eseguito nel topo, al fine di analizzarne l’immunogenicità in vivo. Nonostante siano stati rilevati anticorpi specifici e diretti contro la proteina M1 in un numero statisticamente non significativo di topi saggiati, i dati ottenuti in questo esperimento pilota risultano essere incoraggianti. Infatti, sono stati raggiunti utilizzando VLP prodotte in condizioni di purificazione ancora da ottimizzare. Partendo da questi dati, abbiamo anche generato una serie di costrutti codificanti diversi mutanti della proteina di superficie del virus influenzale HA, accomunati dall’assenza di uno dei suoi domini, ovvero dalla parte globulare (HA-headless). E’ stato, infatti, dimostrato che HA-headless, a differenza della forma wild-type della proteina, è caratterizzata dall’esposizione al sistema immunitario di epitopi altamente conservati e, quindi, rappresenta un’ottima base per lo sviluppo di un vaccino universale, in grado di stimolare la risposta immunitaria verso ceppi virali diversi. Abbiamo analizzato e dimostrato la possibilità di incorporare HAheadless in particelle simil-virali basate sull’espressione della proteina Gag del virus dell’immunodeficienza felina (FIV). Intendiamo ora valutare la possibilità di incorporare HA-headless anche nelle VLP basate su M1 ingegnerizzata e, dopo opportuna purificazione, testare queste ultime e le VLP basate su FIV nel modello murino. In ultimo, al fine di approfondire il ruolo svolto da M2 come sostituta di ESCRT-III nell’ambito della gemmazione del virus dell’influenza, sono stati generati costrutti specifici basati sulla proteina strutturale Gag di FIV, mutata a livello delle sequenze che il nostro gruppo di ricerca ha dimostrato essere essenziali per il reclutamento del pathway di biogenesi degli MVB e per la gemmazione di FIV (Calistri et al., 2009a). I risultati ottenuti suggeriscono che la regione della coda citoplasmatica di M2 è in grado di ripristinare la gemmazione dei mutanti virali. In conclusione, i dati ottenuti in questa tesi di dottorato non solo forniscono importanti informazioni biologiche, relative alle funzioni di due proteine del virus dell’influenza (M1 e M2) e del coinvolgimento di proteine cellulari in fasi essenziali del ciclo replicativo virale, ma pongono interessanti basi per lo sviluppo di nuove strategie vaccinali dirette contro il virus dell’influenza basate su VLP.

Dans cette thèse, l’auto-assemblage en molécules colloïdales de virus en forme de filament, les bactériophages M13, est étudié. Comme première approche, l’affinité de la streptavidine pour la biotine ou un Strep-tag est utilisée et quantitativement comparée. Pour ce faire, des virus modifiés génétiquement, M13-AS, présentant des Strep-tag et des virus M13C7C chimiquement bioconjugués par de la biotine ont réagi via leur extrémité proximale avec des nanoparticules fonctionnalisées par de la streptavidine. Il en résulte la formation de molécules colloïdales en étoile, dont la valence ou nombre de virus par structure, peut être simplement contrôlée par l’excès molaire initial. Cependant, la stabilité de ces molécules colloïdales est limitée par la libération progressive et la dégradation de la streptavidine. Nous avons alors développé une seconde approche basée sur l’affinité soufre-métal, qui s’est avérée à la fois pratique expérimentalement et fiable. Grâce aux groupements disulfures présents sur les cystéines de la protéine P3, des nanoparticules métalliques peuvent se lier à l’extrémité des virus. Le caractère générique de cette méthode est vérifié en faisant varier la nature du métal des nanoparticules ainsi que la souche des virus, dont la sauvage. La valence des structures formées est déterminée en fonction de plusieurs paramètres, dont l’excès molaire initial, la taille des nanoparticules et la force ionique. Un modèle rendant compte des résultats expérimentaux a été élaboré, dont les principales variables sont la surface des nanoparticules et le diamètre effectif électrostatique des virus. Cette approche est étendue à la réalisation de diblocs colloïdaux hétéro bifonctionnels, utilisant les virus comme briques constitutives. Comme preuve de concept, des diblocs bicolores à base de virus sont obtenus par auto-assemblage et leur dynamique est étudiée à l’échelle du bloc élémentaire en microscopie optique de fluorescence. Ainsi, nous avons montré dans cette thèse la réalisation par auto-assemblage d’une nouvelle génération de molécules colloïdales, dont l’auto-organisation peut conduire à la formation de superstructures hiérarchiques hybrides de complexité croissante, potentiellement utiles en sciences des matériaux
In this thesis, the self-assembly of rod-like viral particles, specifically the M13 bacteriophages, into colloidal molecules is studied. As the first method, the affinity of streptavidin to biotin or Strep-tag is used and quantitatively compared. In this case, both biologically engineered M13-AS displaying Strep-tags and chemically biotinylated M13C7C viruses have reacted with streptavidin activated nanoparticles via their functionalized proximal ends. This results in star-like colloidal molecules, whose valency – or number of viruses par structure – can be solely controlled by tuning the initial molar excess. However, the stability of these colloidal molecules is limited by streptavidin release and degradation. Thus, we develop the second method based on the sulfur—metal interactions, which is more convenient and reliable. Thanks to the exposed disulfide groups located at p3 proteins, metallic nanoparticles are able to bind to proximal ends of the M13 virus. The generic feature of this method is verified by using different metals and two virus strains including wt-M13. Afterwards, the control of the valency is explored by varying the initial molar excess, the nanoparticle size and the ionic strength. A quantitative model is built correspondingly, using the surface area of Au nanobead and the effective electrostatic diameter of the virus as variables, which accounts for the assembly of colloidal molecules with desired valencies. This method is further applied to assemble heterobifunctional diblocks by using filamentous viruses as building units. As a proof-of-concept experiment, bicolored diblocks are produced and tracked by each block simultaneously. Overall, we demonstrate the synthesis of a new generation of hybrid colloidal molecules, whose self-organization could serve as a promising means to create novel hierarchical biologic/inorganic superstructures that may find applications in materials science

Human norovirus (NoV) is the leading cause of acute non-bacterial gastroenteritis worldwide. Despite the significant health, emotional, and economic burden caused by human NoV, there are no vaccines or therapeutic interventions for this virus. This is due in major part to the lack of a cell culture system and an animal model for human NoV infection.

Thus, a vector-based vaccine may be ideal for controlling this disease. The major capsid gene (VP1) of a human NoV was inserted into the VSV genome at the glycoprotein (G) and large (L) polymerase gene junction. Recombinant VSV expressing VP1 protein (rVSV-VP1) was recovered from an infectious cDNA clone of VSV. Expression of the capsid protein by VSV resulted in the formation of human NoV virus-like particles (VLPs) that are morphologically and antigenically identical to the native virions. Recombinant rVSV-VP1 was attenuated in cultured mammalian cells as well as in mice. Mice inoculated with a single dose of rVSV-VP1 stimulated a significantly stronger humoral and cellular immune response compared to baculovirus-expressed VLP vaccination. These results demonstrated that that the VSV-based human NoV vaccine induced strong humoral, cellular, and mucosal immunity in a mouse model.

To further improve the safety and efficacy of the VSV-based human NoV vaccine, the gene for the 72kDa heat shock protein (HSP70) was inserted into rVSV and rVSV-VP1 vectors as an adjuvant, which resulted in construction of recombinant VSV expressing HSP70 (rVSV-HSP70) and VSV co-expressing human NoV VP1 protein and HSP70 (rVSV-HPS70-VP1), respectively. At the same inoculation dose, both rVSV-HSP70-VP1 and rVSV-VP1 triggered similar levels of specific immunity, even though VP1 expression by rVSV-HSP70-VP1 was approximately five-fold less than that of rVSV-VP1. To compensate for the reduced VP1 expression levels, the inoculation dose of rVSV-HSP70-VP1 was increased five-fold or same dosage of rVSV-VP1 and rVSV-HSP70 was combined vaccinated. Mice immunized with five does of rVSV-HSP70-VP1 or those receiving combined vaccination generated significantly higher mucosal and/or T cell immunity than those immunized with rVSV-VP1 alone (P<0.05). Therefore, this data indicates that insertion of HSP70 into the VSV vector further attenuates the VSV-based vaccine and HSP70 enhances the human NoV-specific immunities.

To determine whether the VSV-based human NoV vaccine confers protection from human NoV challenge, a gnotobiotic pig model was developed. Newborn gnotobiotic piglets vaccinated intranasally with rVSV-based vaccine (rVSV-VP1) produced high levels of specific serum IgG and fecal and vaginal IgA antibody. Three weeks after vaccination, piglets were orally challenged with human NoV. All three piglets in the unvaccinated challenged group developed histopathologic lesions typical of human NoV infection in the duodenum and proximal jejunum on day 5 post-challenge. However, only one of five vaccinated piglets exhibited focal epithelium loss and villous atrophy, and mild edema in the small intestines. Immunofluorescent assay showed that a large amount of human NoV antigens were detected in duodenum, jejunum, and ileum of the challenge control group but not vaccinated group. These results demonstrate that the rVSV-based human NoV vaccine triggered partially protective immunity in swine and protected gnotobiotic pigs from challenge by human NoV.

Viruses are entities which are made of a few genes and are reliant on obligate parasitism to propagate. Due to the obligate connection to their hosts, virus evolution is constrained to the type of host. Viruses however do transmit to evolutionary distinct hosts; in these cases, the phylogenetic relationship of the hosts usually are close. In some instances, RNA-viruses have made host jumps between evolutionary distant hosts, such as the host jump from invertebrates to vertebrates, and fungi to arthropod. Partitiviruses are double stranded RNA viruses which mainly infect fungi and plants. The defining characteristic of these double stranded RNA viruses are the double layered capsids which are formed by a single open reading frame (ORF). The capsid proteins form icosahedral virus particles which are in the magnitude of 30-40 nm. Metagenomic studies have discovered partitiviruses originating from an insect in the Odanata family, a finding which contradicts the fungal host specificity of partitiviruses. The finding of the Hubei.PLV 11 thus implies the existence of a partitiviruses containing structural elements in their capsids which could be involved in the infection of arthropods. Thus, this virus could be used as a model for a structural comparison with its fungi infecting relatives with hopes to identify common viral structural factors necessary for the infection of arthropods. For this purpose, the Hubei.PLV ORF was cloned and then transfected into insect Spodoptera frugiperda (Sf-9) cells using a baculovirus expression system, “bac-to-bac” expression system. The FLAG-tagged capsid proteins were expressed by the Sf-9 cells to be approximately 60 kDa. After ultra-centrifugation in a sucrose gradient, some spontaneous assembly into the expected ~40 nm icosahedral virus-like particles were observed using low resolution scanning electron microscopy. The observed particles were also confirmed by a dynamic light scattering experiment (DLS) and a higher resolution cryo-EM microscope. Thus, the bac-to-bac expression system can be used to produce VLPs from this genus of viruses, and this metagenomically derived virus genome. However, for future success in defining a high-resolution model of this virus, it is recommended that the Sf-9 culture volume is sufficiently high for enough particle production which is necessary for a high-resolution map. The other virus, the Southern pygmy squid Flavilike virus (SpSFV) has been suggested to be the oldest relative of the land based flaviviruses. The SpSFV was found to be the most divergent of the flaviviruses, and to infect invertebrates. Solving for the structure of the SpSFV and comparing it to vertebrate infecting flaviviruses could therefore lead to the identification of factors necessary for the adaptation to vertebrates and thus the humoral immunity by flaviviruses. The soluble E-protein was expressed using the bac-to-bac expression system. The protein was indicated to be multiglycosylated and approximately 50 kDa which is in line with other strains in the genus. Affinity chromatography did not elute this protein, likely due to the His-tag not being spatially available. Cation exchange could elute some protein, but not much from the small ~30 mL culture. To conclude, VLP assembly was confirmed by the Hubei.PLV, thus, solving for the structure is a distinct possibility when a larger Sf-9 culture is used to produce the VLPs. For the SpSFV soluble E-protein, the protein is secreted into the supernatant of the Sf-9 cultures, making purification a possibility. For this, a large Sf-9 culture can be used to produce this protein and then purify it with a cat-ion exchange chromatography.

A doença de Chagas, também denomianada de Tripanossomíase Americana, é uma infeção causada pelo protozoário flagelado Trypanosoma cruzi. Foi estimado que 7 milhões de pessoas do mundo estão infetadas com T. cruzi e mais de 25 milhões estão em risco de infeção. Ao longo dos anos têm sido testados vários antigénios com o intuito de produzir vacinas contra a doença de Chagas e até o presente nenhuma destas conseguiu chegar a ensaios clínicos em humanos, embora tenham sido aplicadas várias estratégias imunoterapêuticas para combater a infeção, tais como vacinas de DNA e antigénios recombinantes. A produção de vacinas baseadas nas Virus-like Particles (VLPs) ainda não foi completamente explorada no contexto da doença de Chagas. Neste trabalho pretendeu-se avaliar a resposta imune humoral em murganhos imunizados com VLPs de Triatoma virus (TrV-VLPs) que contêm peptídeos com sequências antigénicas de T. cruzi. Começou-se por caracterizar as propriedades imunoestimuladoras de TrV-VLPs através da otimização do processo de imunização. Por fim, definiu-se a resposta humoral nos murganhos avaliando a produção dos anticorpos anti-TrV-VLPs e anticorpos anti-T. cruzi. Foi selecionado o peptídeo sintético SATA-TLQPVERVL baseado no trabalho realizado por outros investigadores. Neste estudo, o peptídeo foi quimicamente conjugado à superfície das TrV-VLPs por meio dos ligantes com grupos específicos, posteriormente, foi submetido a ensaios de imunização em murganhos. Inicialmente, não foi possível verificar a indução de anticorpos IgG totais anti-peptídeo nos animais imunizados com TrV-VLP conjugado com peptídeo. Posteriormente, alterou-se a estratégia de produção do peptídeo, onde se adicionou uma D-ala para melhorar a sua biodisponibilidade. Com esta nova estratégia foi possível determinar a presença de anticorpos IgG totais anti-SATA-TLQPVERVL-D-ala, bem como as subclasses, IgG1 e IgG2a, nos murganhos imunizados. Em conclusão, os resultados finais sugerem que o peptídeo SATA-TLQPVERVL-D-ala conjugado à superfície da TrV-VLPS poderá ser um importante candidato para o desenvolvimento de potenciais vacinas para a doença de Chagas.
Chagas disease, also known as American trypanosomiasis, is a tropical parasitic disease caused by the protozoan Trypanosoma cruzi. It was estimated that 7 million people are infected with T. cruzi and over 25 million are at risk of infection. Over the years, several antigens have been tested with the goal of vaccine production against Chagas disease, but neither was able to reach human clinical trials. In an effort to revert this situation, various immunotherapeutic strategies were applied to try and oppose the spreading of the infection, namely the DNA vaccines, and recombinant antigens. Production of vaccines based on Virus-like Particles (VLPs) has not yet been explored on the context of Chagas desease. This work proposes to evaluate the humoral immunity on house mice (Mus musculus) immunized with VLPs of Triatoma virus (TrV-VLPs) that contain peptides with antigenic sequences of T. cruzi. The first step was to characterize the immunostimulant properties of TrV-VLPs by optimizing the immunization process. Then, humoral response on house mice was defined evaluating the production of antibodies anti-VLPs and antibodies anti-T. cruzi. The synthetic peptide (SATA-TLQPVERVL), based on work done by others, was selected. In this study, the peptide was chemically conjugated on the surface of TrV’s VLP via ligands with specific groups and posteriorly submitted to trials of immunization in house mice. Initially, it wasn’t possible to verify the production of total IgG antibodies anti-peptide on the animals immunized with TrV’s VLP conjugated with the peptide. Posteriorly, by changing the strategy of peptide production, where D-ala was added to improve its bioavailability. Following this new strategy, it was possible to detect the presence of total IgG antibodies anti-SATA-TLQPVERVL-D-ala with the associated subclasses, IgG1 and IgG2a. In conclusion, final results suggest that this peptide, SATA-TLQPVERVL-D-ala conjugated on the surface of TrV’s VLP might well be is an important candidate to the development of potential vaccines for Chagas desease.