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Investigating alternative delivery systems for self-Amplifying RNA vaccines
Get PDFThe rabies virus is an enveloped, single stranded, negative-sense RNA virus of the Lyssavirus genus, zoonotic pathogens within the family Rhabdoviridae. Although extensive effort has been made in the last decades to develop efficacious vaccines to prevent rabies spread, the virus is still responsible for the mortality of about 24,000 to 90,000 people per year especially in developing countries and it has been classified as one of the major causes of death from infectious diseases in humans.;Commercially available rabies vaccines for humans are considered effective, however the production costs are very high and multiple injections are required to achieve protection. Therefore, the development of new vaccines to reduce the toll of rabies disease in the developing world would be highly desirable. Within this project a nucleic acid based vaccine strategy - in particular self-amplifying RNA vaccine (SAM)- has been investigated since this platform was previously reported to elicit protective immune responses, particularly in the case of cell-mediated responses in a safe manner and for a variety of virus disease.;To enhance biological stability and cell internalisation, SAM was combined with four cationic delivery systems. Oil-in-water cationic nano emulsions (CNE), polymeric nanoparticles (NPs), lipid nanoparticles (SLNs) and liposomes were formulated in the absence of or in combination with a specific SAM vaccine. Despite the differences in formulation composition, all samples contained the same concentration of cationic lipid - 1, 2-dioleoyl-3-trimethylammonium-propane (DOTAP), or dimethyldioctadecylammonium (DDA) - known as immunostimulants.;In the preliminary studies, two different manufacturing processes such as Microfluidics and Microfluidisation were applied. As a proof of concept, anionic liposomes and solid lipid nanoparticles were formulated and ovalbumin was encapsulated within the delivery systems as model protein antigen. Resulting carriers were compared in terms of their physico-chemical properties. The purpose was to obtain homogeneous formulations with a diameter in the nanometres range with a given manufacturing method. Furthermore, dialysis, tangential flow filtration (TFF) and size exclusion chromatography (SEC) have been tested as purification methods and compared in terms of the ability to remove both residual organic solvent and unloaded protein from samples without altering physico-chemical attributes.;These process parameters and purification method optimisations were then applied to produce cationic CNE, NPs, SLNs and liposomes in combination with a specific SAM vaccine. In the preliminary studies and during formulations development optimisation, SAM encoding for green fluorescent protein (SAM-GFP) was used as a model SAM with a reporter function,given the ease of detection in in-vitro cell cultures. However, SAM encoding for rabies glycoprotein (SAM-Rabies) represented the actual antigen of interest, employed in this project for further in vivo analysis. Cationic SLNs, NPs and liposomes were produced using microfluidics, since this method required smaller volumes compared to the Microfluidisation, thus avoiding waste of reagents.;However, the Microfluidizer was used to reduce CNE size,due to incompatibility between CNE component and microfluidics chip. Moreover, particles were formulated with SAM encoding the antigen of interest and loaded into or adsorbed onto cationic carriers. All delivery systems were evaluated according to their physico-chemical properties: hydrodynamic radius, sample homogeneity (polydispersity index - PDI) and surface charge. Furthermore, in vitro activity was investigated using three different cell lines:bone marrow derived macrophages (BMDM), bone marrow derived dendritic cells (BMDC) and baby hamster kidney cells (BHK).;SAM uptake and antigen expression from each formulation in each cell line were used to discriminate and down-select formulations for invivo studies. In the in vivo studies, biodistribution of carriers alone or in combination with SAM were performed. Briefly the selected SAM-carriers were administered intramuscularly (i.m.) to BALB/c mice and their movement in the animal body was tracked using a radiolabelling technique thereby allowing measurement of formulations at chosen time-points and in specific organs. The aim of the study was to understand the pharmacokinetic profile of formulations in a mouse model and assess whether biodistribution might correlate with subsequent immunogenicity studies.;The initial attempt of these studies was to (i) find the antigen dose to induce high antibody and cellular responses in vivo and (ii) to compare the adjuvant properties of selected cationic candidates (i.e. SAM encapsulating DOTAP NPs, DOTAP liposomes and DDA liposomes) after i.m. injection. Formulations were selected according to the potency of inducing antigen expression in vitro. The commercial vaccine Rabipur, which is an inactivated virus rabies vaccine, was used as comparator. The aim was to find a valid and more cost-effective alternative formulation which induced an immune response comparable or superior to the commercial vaccine.;Data showed that DOTAP NPs were the most potent in triggering IgG titers among candidates and the antibody levels were equivalent to the ones induced by the commercial vaccine after a single dose. Interestingly, the GMT was well above the protective threshold despite the antigen dose used, thus meaning that elicited antibodies were functional against rabies glycoprotein G. In terms of cellular response all candidates were able to activate both CD4+ and CD8+ T cells in a comparable manner to the vaccine on the market.;Moreover, to evaluate if changing the route of administration might affect carriers' potency,SAM encapsulating candidates were also administered intradermally (i.d.) and intranasally (i.n.), and formulations immunogenicity was evaluated according to IgG titres and cellular response. To do so, DOTAP NPs and DOTAP SLNs were selected; NPs were tested considering the promising outcome from the first in vivo study, whereas SLNs were introduced although poor in vitro antigen expression. The aim was to understand the power of in vitro models to predict in vivo antigen immunogenicity.;Results highlighted that SLNs injected i.m.showed increased immunogenicity compared to both NPs and the licenced vaccine after a single dose. Moreover, the potency of SLNs was also seen after intradermal administration,where SLNs were as potent as Rabipur to elicit IgG titer in mice after two vaccinations, inducing comparable innate and adaptive immunity to the vaccine on the market. Herein it was also reported that two doses of SAM SLNs injected i.n. induced a humoral immunity which was higher than the one elicited by Rabipur.;Interestingly, intranasal administration of SLNs led to a higher percentage of IL-2 producing antigen specific CD4+ T cells compared to the licenced in both spleens and lungs. Although a significant difference was observed among formulations in the ability to enhance antigen-specific IgG titres, immunogenicity did not directly correlate with biodistribution, where carriers' pharmacokinetics were indeed similar. All together, these findings are encouraging and demonstrate that coformulation of SAM vaccine and solid lipid nanoparticles might be a valid and more advantageous alternative to produce rabies vaccines, with augmented patient' safety and compliance.The rabies virus is an enveloped, single stranded, negative-sense RNA virus of the Lyssavirus genus, zoonotic pathogens within the family Rhabdoviridae. Although extensive effort has been made in the last decades to develop efficacious vaccines to prevent rabies spread, the virus is still responsible for the mortality of about 24,000 to 90,000 people per year especially in developing countries and it has been classified as one of the major causes of death from infectious diseases in humans.;Commercially available rabies vaccines for humans are considered effective, however the production costs are very high and multiple injections are required to achieve protection. Therefore, the development of new vaccines to reduce the toll of rabies disease in the developing world would be highly desirable. Within this project a nucleic acid based vaccine strategy - in particular self-amplifying RNA vaccine (SAM)- has been investigated since this platform was previously reported to elicit protective immune responses, particularly in the case of cell-mediated responses in a safe manner and for a variety of virus disease.;To enhance biological stability and cell internalisation, SAM was combined with four cationic delivery systems. Oil-in-water cationic nano emulsions (CNE), polymeric nanoparticles (NPs), lipid nanoparticles (SLNs) and liposomes were formulated in the absence of or in combination with a specific SAM vaccine. Despite the differences in formulation composition, all samples contained the same concentration of cationic lipid - 1, 2-dioleoyl-3-trimethylammonium-propane (DOTAP), or dimethyldioctadecylammonium (DDA) - known as immunostimulants.;In the preliminary studies, two different manufacturing processes such as Microfluidics and Microfluidisation were applied. As a proof of concept, anionic liposomes and solid lipid nanoparticles were formulated and ovalbumin was encapsulated within the delivery systems as model protein antigen. Resulting carriers were compared in terms of their physico-chemical properties. The purpose was to obtain homogeneous formulations with a diameter in the nanometres range with a given manufacturing method. Furthermore, dialysis, tangential flow filtration (TFF) and size exclusion chromatography (SEC) have been tested as purification methods and compared in terms of the ability to remove both residual organic solvent and unloaded protein from samples without altering physico-chemical attributes.;These process parameters and purification method optimisations were then applied to produce cationic CNE, NPs, SLNs and liposomes in combination with a specific SAM vaccine. In the preliminary studies and during formulations development optimisation, SAM encoding for green fluorescent protein (SAM-GFP) was used as a model SAM with a reporter function,given the ease of detection in in-vitro cell cultures. However, SAM encoding for rabies glycoprotein (SAM-Rabies) represented the actual antigen of interest, employed in this project for further in vivo analysis. Cationic SLNs, NPs and liposomes were produced using microfluidics, since this method required smaller volumes compared to the Microfluidisation, thus avoiding waste of reagents.;However, the Microfluidizer was used to reduce CNE size,due to incompatibility between CNE component and microfluidics chip. Moreover, particles were formulated with SAM encoding the antigen of interest and loaded into or adsorbed onto cationic carriers. All delivery systems were evaluated according to their physico-chemical properties: hydrodynamic radius, sample homogeneity (polydispersity index - PDI) and surface charge. Furthermore, in vitro activity was investigated using three different cell lines:bone marrow derived macrophages (BMDM), bone marrow derived dendritic cells (BMDC) and baby hamster kidney cells (BHK).;SAM uptake and antigen expression from each formulation in each cell line were used to discriminate and down-select formulations for invivo studies. In the in vivo studies, biodistribution of carriers alone or in combination with SAM were performed. Briefly the selected SAM-carriers were administered intramuscularly (i.m.) to BALB/c mice and their movement in the animal body was tracked using a radiolabelling technique thereby allowing measurement of formulations at chosen time-points and in specific organs. The aim of the study was to understand the pharmacokinetic profile of formulations in a mouse model and assess whether biodistribution might correlate with subsequent immunogenicity studies.;The initial attempt of these studies was to (i) find the antigen dose to induce high antibody and cellular responses in vivo and (ii) to compare the adjuvant properties of selected cationic candidates (i.e. SAM encapsulating DOTAP NPs, DOTAP liposomes and DDA liposomes) after i.m. injection. Formulations were selected according to the potency of inducing antigen expression in vitro. The commercial vaccine Rabipur, which is an inactivated virus rabies vaccine, was used as comparator. The aim was to find a valid and more cost-effective alternative formulation which induced an immune response comparable or superior to the commercial vaccine.;Data showed that DOTAP NPs were the most potent in triggering IgG titers among candidates and the antibody levels were equivalent to the ones induced by the commercial vaccine after a single dose. Interestingly, the GMT was well above the protective threshold despite the antigen dose used, thus meaning that elicited antibodies were functional against rabies glycoprotein G. In terms of cellular response all candidates were able to activate both CD4+ and CD8+ T cells in a comparable manner to the vaccine on the market.;Moreover, to evaluate if changing the route of administration might affect carriers' potency,SAM encapsulating candidates were also administered intradermally (i.d.) and intranasally (i.n.), and formulations immunogenicity was evaluated according to IgG titres and cellular response. To do so, DOTAP NPs and DOTAP SLNs were selected; NPs were tested considering the promising outcome from the first in vivo study, whereas SLNs were introduced although poor in vitro antigen expression. The aim was to understand the power of in vitro models to predict in vivo antigen immunogenicity.;Results highlighted that SLNs injected i.m.showed increased immunogenicity compared to both NPs and the licenced vaccine after a single dose. Moreover, the potency of SLNs was also seen after intradermal administration,where SLNs were as potent as Rabipur to elicit IgG titer in mice after two vaccinations, inducing comparable innate and adaptive immunity to the vaccine on the market. Herein it was also reported that two doses of SAM SLNs injected i.n. induced a humoral immunity which was higher than the one elicited by Rabipur.;Interestingly, intranasal administration of SLNs led to a higher percentage of IL-2 producing antigen specific CD4+ T cells compared to the licenced in both spleens and lungs. Although a significant difference was observed among formulations in the ability to enhance antigen-specific IgG titres, immunogenicity did not directly correlate with biodistribution, where carriers' pharmacokinetics were indeed similar. All together, these findings are encouraging and demonstrate that coformulation of SAM vaccine and solid lipid nanoparticles might be a valid and more advantageous alternative to produce rabies vaccines, with augmented patient' safety and compliance
microfluidic method to manufacture protein.loaded nanocarriers: process paramters analyis
No full textGoing Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
Author-wise bibliometric analysis based on entropy.
No full textAuthor-wise bibliometric analysis based on entropy.</p
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