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Construction of a modified live HP-PRRS virus vaccine and an attenuated listeria vaccine vector using reverse geneticsRen, Jie January 1900 (has links)
Master of Science / Department of Anatomy and Physiology / Jishu Shi / The development of reverse genetics systems for the manipulation of viral and bacterial genomes has provided platforms for identifying virulence genes, studying pathogenesis and developing vaccines. Replication-competent vaccines (e.g., modified live virus (MLV) vaccines and replicating viral/bacterial vectors) are considered the most efficacious approach for vaccine development. We constructed replication-competent candidate vaccines for two viral diseases in pigs via reverse genetics. The first vaccine we designed is to protect against highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV). HP-PRRSV can cause high mortality in pigs of all ages. Vaccines to protect pigs from HP-PRRSV are not commercially available in the US. According to previous studies, the non-structural protein (NSP) coding region of HP-PRRSV is closely related to the high mortality rate and the structural protein (SP) coding region contributes to the induction of broadly protective neutralizing antibodies. We created a chimeric PRRSV, of which the SP coding region was derived from HP-PRRSV and NSP coding region was derived from a low-pathogenic strain. This chimeric PRRSV caused similar CPE in cells as parental viruses, but had slower growth kinetics. We hypothesize that this chimeric virus will have a low pathogenicity and could serve as a candidate vaccine that can provide protection against HP-PRRV. The second vaccine vector is a modified Listeria innocua (L.inn), a non-pathogenic strain of Listeria. Genetically related Listeria monocytogenes (L.m) is a well-known intracellular pathogen that encodes specialized virulent determinants facilitating its intracellular growth and spread. Our goal is to make L.inn a vaccine vector that can deliver classical swine fever (CSF) viral antigen into intracellular environments by complementation of L.inn with selected L.m virulence genes necessary for intracellular survival and induction of a robust immune response. In this study, we constructed a shuttle vector pHT-E2 that can express CSFV antigen E2 in L.inn. We cloned the plcA-prfA operon of L.m virulence gene cluster (vgc) into pHT-E2, which enhanced the expression of E2 in L.inn. In future studies, we plan to clone additional L.m virulence genes into the shuttle vector to increase immunogenicity of this recombinant L.inn and test its ability to protect pigs from CSFV.
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Modeling future range expansion and management strategies for an invasive squirrel speciesGoldstein, Emily A., Butler, Fidelma, Lawton, Colin 18 February 2016 (has links)
Successful management of an invasive
species requires in depth knowledge of the invader, the
invaded ecosystem, and their interactions. The complexity
of the species-system interactions can be
reduced and represented in ecological models for
better comprehension. In this study, a spatially explicit
population model was created using the RAMAS
software package to simulate the past and future
invasion dynamics of the eastern grey squirrel (Sciurus
carolinensis) in the fragmented habitat in case study
areas in Ireland. This invasive squirrel species causes
economic damage by bark stripping forest crops and is
associated with the decline of its native congener (S.
vulgaris). Three combinations of demographic and
dispersal parameters, which best matched the distribution
of the species shortly after introduction, were
used to simulate invasion dynamics. Future population
expansion was modeled under scenarios of no control
and two different management strategies: fatal culls
and immunocontraceptive vaccination programmes.
In the absence of control, the grey squirrel range is
predicted to expand to the south and southwest of
Ireland endangering internationally important habitats,
vulnerable forest crops, and the native red
squirrel. The model revealed that region-wide intensive
and coordinated culls would have the greatest
impact on grey squirrel populations. Control strategies
consisting solely of immunocontraceptive vaccines,
often preferred by public interest groups, are predicted
to be less effective. Complete eradication of the grey
squirrel from Ireland is not economically feasible and
strategic evidence-based management is required to
limit further range expansion. Ecological models can
be used to choose between informed management
strategies based on predicted outcomes.
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Evaluation of Aspergillus as a host for the production of viral proteins using hepatitis B as a modelPluddemann, Annette, 1972- 12 1900 (has links)
Dissertation (PhD)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Since the advent of recombinant DNA technology in the 1970s, various
microbial hosts have been employed for the efficient high-level heterologous
production of a variety of proteins, ranging from enzymes and reagents to
therapeutics and vaccines. More recent microbial hosts to be employed for
these purposes are filamentous fungi, and particularly the genus Aspergillus.
Aspergilli have been associated with industrial processes for many years and
are used in the production of antibiotics, enzymes, citric acid and Oriental foods
and beverages, and thus strains such as Aspergillus niger and Aspergillus
oryzae have been afforded GRAS (Generally Regarded 8s ~afe) status. They
also secrete copious amounts of homologous and heterologous proteins and
are able to perform post-translational modifications effectively. Various proteins
of pharmaceutical interest have been successfully expressed in Aspergillus, but
the potential of these fungi to produce heterologous viral proteins has not been
explored extensively.
In this study, we evaluated the potential of the filamentous fungi A. niger and
Aspergillus awamori as alternative hosts for the heterologous production of
hepatitis B viral proteins. Hepatitis B is a serious, potentially lethal liver disease
that affects 2000 million people world-wide and has a high endemicity in
Southern Africa. Currently there is no effective treatment for viral hepatitis and
thus a mass vaccination strategy is the only solution to curb the spread of the
disease. This kind of vaccination strategy requires a cheap, safe and effective
vaccine and these objectives have been achieved in the development of
recombinant subunit vaccines from yeasts such as Saccharomyces cerevisiae,
Hansenula polymorpha and Pichia pastoris that are commercially available.
The hepatitis B virus envelope consists of a membrane fraction and three
proteins, namely the major (S) protein (encoded by the S gene), the middle (M)
protein (encoded by the preS2S gene) and the large (L) protein (encoded by the
preS1preS2S gene). When produced in the above-mentioned yeasts, the S
protein was shown to spontaneously assemble into pseudoviral particles devoid
of viral DNA, which were then purified and used as vaccine. In the present study the Sand preS1preS2S genes from a local isolate of
hepatitis B subtype adw2 were placed under transcriptional control of the
constitutive Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase
(gpdA) promoter and the inducible A. niger glucoamylase (glaA) promoter. The
respective viral genes were also fused to the region encoding the catalytic
domain of the highly expressed and secreted A. niger glucoamylase, which
served as a carrier moiety to possibly facilitate viral protein secretion. The
various gene constructs were subsequently transformed to laboratory strains of
A. niger and A. awamori and numerous transformants were obtained. One
A. niger transformant carrying the S gene under control of the gpdA promoter
contained approximately 7 integrated copies of the expression cassette and
produced hepatitis B pseudoviral particles intracellularly at levels of 0.4 mg/I
culture. These levels are approximately ten-fold higher than those initially
obtained from the yeast S.cerevisiae, which showed yields of 0.01 to
0.025 mg/I. None of the other transformants could be shown to produce
recombinant S or L protein and no secretion of viral protein could be
demonstrated. This could be attributed to numerous factors, including vector
copy number, site of integration or proteolytic activity. The most important
insight emerging from this work regarding secretion of heterologous viral protein
was that the addition of a carrier protein hampered, rather than enhanced
secretion of the viral envelope protein, due to the inherent properties of viral
protein assembly.
This work also serves as a "proof of principle", showing that Aspergillus is
indeed a viable alternative host for the production of hepatitis B pseudoviral
particles, and could be investigated further for its potential as host for the
heterologous expression of other viral proteins. / AFRIKAANSE OPSOMMING: Sedert die ontwikkeling van rekombinante DNA tegnologie in die sewentigerjare
is verskeie mikroorganismes reeds gebruik vir die doeltreffende produksie van
'n verskeidenheid proteïne teen hoë vlakke; onder andere ensieme, reagense,
terapeutiese middels en vaksiene. Onlangs is filamentagtige swamme, veral
van die genus Aspergillus, ontwikkel vir heteroloë proteïenproduksie. Aspergilli
word al vir baie jare in nywerheidsprosesse gebruik, onder andere in die
vervaardiging van antibiotika, ensieme, sitroensuur en sekere Oosterse
voedsel- en drankprodukte. As gevolg van hierdie jarelange gebruik van rasse
soos Aspergillus niger en Aspergillus oryzae, word hulle algemeen aanvaar as
veilig vir menslike gebruik. Hierdie swamme besit veral die vermoë om hoë
vlakke van homoloë en heteroloë proteïene uit te skei en die na-translasiemodifisering
van proteïene korrek uit te voer. Verskeie proteïene van
farmaseutiese belang is al suksesvol in Aspergillus uitgedruk, maar die
potensiaal van hierdie swamme om virale proteïene te vervaardig is nog nie
deeglik ondersoek nie.
Hierdie studie ondersoek die geskiktheid van die filamentagtige swamme
A. niger en Aspergillus awamori om as alternatiewe gashere vir die heteroloë
produksie van hepatitis B proteïene te dien. Hepatitis B is 'n ernstige en selfs
dodelike lewersiekte. Omtrent 2000 miljoen mense wêreld-wyd is met die virus
geïnfekteer en dit is veral endemies in Suiderlike Afrika. Daar is tans geen
doeltreffende behandeling vir virale hepatitis en dus is wêreld-wye
inentingsprogramme die enigste oplossing om die verspreiding van die siekte te
bekamp. Hierdie inentingsstrategie vereis die beskikbaarheid van 'n
bekostigbare, veilige en doeltreffende vaksien. Die rekombinante subeenheidvaksiene
wat ontwikkel is deur van gashere soos Saccharomyces cerevisiae,
Hansenula polymorpha en Pichia pastoris gebruik te maak, voldoen aan hierdie
vereistes en is kommersieel beskikbaar. Die omhulsel van die hepatitis B virus
bestaan uit 'n membraangedeelte en drie proteïene, naamlik die hoofproteïen
(S) (gekodeer deur die S-geen), die middelproteïen (M) (gekodeer deur die
preS2S-geen) en die grootproteïen (L) (gekodeer deur die preS1preS2S-geen). Wanneer die S-proteïen in bo-genoemde giste uitgedruk word, vorm dit
spontaan pseudovirale partikels wat nie virale DNA bevat nie. Hierdie partikels
word dan gesuiwer en as vaksien gebruik.
In hierdie studie is die S- en preS1preS2S-gene, vanaf 'n plaaslike isolaat van
hepatitis B subtipe adw2, onder transkripsionele beheer van die konstitutiewe
Aspergillus nidulans gliseraldehied-3-fosfaat-dehidrogenasepromoter (gpdA) en
die induseerbare A. niger glukoamilasepromoter (glaA) geplaas. Die
onderskeie virale gene is ook aan die koderende gedeelte vir die katalitiese
domein van A. niger glukoamilase gelas om fusieproteïene te vorm.
Glukoamilase word teen hoë vlakke deur Aspergillus vervaardig en uitgeskei en
kan dus moontlik dien as draerproteïen om sekresie van die proteïne te
bevorder. Transformasie van die geenkonstrukte na laboratoriumrasse van
A. niger en A. awamori het verskeie transformante gelewer. Een A. niger
transformant bevattende die S-geen onder transkripsionele beheer van die
gpdA promoter het minstens sewe kopieë van die uitdrukkingskaset in sy
genoom geïntegreer en het hepatitis B pseudovirale partikels intrasellulêr teen
vlakke van 0.4 mg/I swamkultuur vervaardig. Hierdie vlakke is omtrent tienvoudig
hoër as die vlakke van 0.01 - 0.025 mg/I wat S.cerevisiae oorspronklik
opgelewer het. Nie een van die ander transformante het rekombinante S of L
proteïene vervaardig nie en sekresie van virale proteïen kon nie getoon word
nie. Hierdie verskynsel mag te wyte wees aan verskeie faktore insluitende
vektor-kopiegetal, setel van integrasie en proteolitiese aktiwiteit. Die
belangrikste insig uit hierdie studie aangaande sekresie van heteroloë virale
proteïene is dat die koppeling van die virale omhulsel-proteïen aan 'n
draerproteïen sekresie benadeel het, eerder as om dit te bevorder. Hierdie
verskynsel is te wyte aan die inherente geneigdheid van virale omhulselproteïene
om 'n kompleks te vorm.
Die studie dien ook as "bewys van beginsel" dat Aspergillus wel 'n werkbare
alternatiewe gasheer vir die produksie van hepatitis B pseudovirale partikels is,
en dat dit verder ondersoek sou kon word as potensiële gasheer vir die
heteroloë uitdrukking van ander virale proteïene.
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Investigating the use of gold nanoparticles in vaccine deliveryGregory, Anthony Edward January 2013 (has links)
Vaccination is one of the most effective public health interventions in the world, saving millions of lives and preventing the onset of debilitating diseases. With widespread emergence of multi-drug resistant pathogens, the importance of preventative medicine has become even more apparent. However, one of the limiting factors in developing novel vaccines that are both safe and highly immunogenic is the availability of adjuvant delivery systems licensed for human use. The purpose of this study was to investigate the role gold nanoparticles could play as an effective vaccine delivery system. A variety of coupling chemistries were explored for their ability to conjugate protein and polysaccharide antigens onto the surface of gold nanoparticles for the development of vaccines against a number of biologically important human pathogens including Y. pestis, B. mallei and S. pneumoniae. Retention of antigenicity and coupling efficiency of conjugated molecules was measured using characterisation techniques such as localised surface plasmon resonance and immunoblotting. Gold nanoparticle coupled antigens were then used to immunise mice and to measure the protective efficacy and the immunological response induced. The findings indicate antigen-specific immune responses are elevated when an antigen is coupled onto gold nanoparticles. Moreover, immunological data from nanoparticle coupled glycoconjugate vaccines against B. mallei and S. pneumoniae indicate the likely presence of a strong T cell immune response which is essential for providing immunological memory. Finally, an intracellular trafficking assay was carried out to identify some of the mechanisms that might be involved in uptake of gold nanoparticles into professional phagocytes. Confocal imaging of receptors associated with endosomal compartments revealed that gold nanoparticles may enter cells through multiple pathways. The findings reported in this study suggest that gold nanoparticles may be an excellent candidate for further investigation as a novel vaccine delivery system.
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Normalization and Informed Decision-making in Public Health Programs: A Case Study of HPV Vaccination in CanadaNavaneelan, Tanya 19 November 2012 (has links)
This thesis examined the evidence, policy decision-making, and implementation of HPV vaccination in Canada as a case study to explore normalization versus individualized decision making in public health programs. Mixed methods were used: a systematic review, content analyses and policy document analysis.
Overall, the scientific evidence supported an effect of vaccination against HPV infection and precancerous cervical lesions, but evidence regarding cervical cancer incidence or mortality is lacking. Scientific and medical communities appeared optimistic about the vaccine, but cautious about its readiness for routine implementation. Policy decision-making was initially cautious, but shifted towards active program implementation, possibly related to the availability of federal funding. The educational materials and media coverage both sent clearly normalizing messages about HPV vaccination.
The discussion suggests that HPV vaccination might be more suited to an individualized than population approach, but many factors coincided to promote its implementation, in Canada, within a traditional public health model.
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The design and development of an HIV-1 vaccine to elicit a broadly neutralising antibody responseWan, Lai Kin Derek January 2012 (has links)
Despite 30 years of research, a prophylactic vaccine against HIV-1 is still lacking and is urgently needed in order to control the global AIDS pandemic. The discovery of broadly neutralising antibodies (BNAbs) was an important step for HIV-1 research but no vaccine candidate tested so far has been able to reproduce responses containing such antibodies, and it remains unclear how this could be achieved via immunisation. In this thesis, I attempted to explore this gap of knowledge in two ways. First, certain features (‘signatures’) of the Env protein that were associated with a broadly neutralising response were identified through machine learning. Further characterisation of these signatures revealed several ways by which these naturally-occurring mutations might alter the immunogenicity of the Env protein that could result in the elicitation of a broadly neutralising response. The incorporation of such signatures in future vaccine design could be useful as the Env protein might adopt a conformation that encourages the elicitation of a broadly neutralising response. Second, 3 novel vaccination approaches were proposed aiming to induce a BNAb antibody response. The development of 2 approaches proved to be difficult and was not continued. For the third approach, non-neutralising immunogen-derived antibodies were used to mask immunodominant epitopes on the Env protein (i.e. ‘antibody-shielding’), thus allowing the antibody response to be focused to the highly conserved CD4 binding site (CD4bs). Subsequent immunisation of the antibody-shielded gp120 proteins in mice and rabbits demonstrated that antibody-shielding was able to significantly dampen the V3-specific antibody response while retaining the CD4bs-specific response. However, the antibody response to the V1/V2 loop was enhanced upon V3-dampening which indicates that further optimisation of the antibody-shield is needed in order to eliminate any antibody response towards the immunodominant regions. In conclusion, these results are the first description of a number of novel vaccination ideas and provide valuable insights into how these approaches could be optimised to become effective HIV-1 vaccines that can lead to the elicitation of a broadly neutralising antibody response.
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Investigation of the potential of PorA and FetA as meningococcal vaccine componentsSanders, Holly January 2012 (has links)
In the search for a vaccine providing comprehensive protection against meningococcal disease, one vaccine currently under development contains the immunogenic proteins PorA and FetA in meningococcal outer membrane vesicles (OMVs). To achieve high levels of coverage against disease-causing isolates, the antigenic variability of these proteins could be overcome using knowledge of meningococcal epidemiology and population structure. In this study, the possible implications of variable expression levels of PorA and FetA on vaccine efficacy were investigated. Producing OMVs containing consistent amounts of FetA is difficult due to iron-repressed expression; therefore, meningococcal strains were constructed which constitutively expressed FetA at increased levels for OMV vaccine production and analysis. In mice, OMVs from modified strains induced antibodies against both PorA and FetA. These antibodies acted synergistically in a serum bactericidal assay; however, antibodies against FetA were weakly bactericidal alone. The potential to increase levels of PorA- and FetA-specific bactericidal antibodies with a prime-boost strategy, using OMV and protein inoculums, was also tested. While successful for a weakly-immunogenic PorA variant, a similar strategy did not increase bactericidal activity against FetA. Although antibodies against FetA can be induced following OMV immunisation, sufficient antigen expression in target bacteria is also required for bactericidal killing; therefore, the variability and regulation of porA and fetA transcription was investigated in a range of isolates. Despite differences in regulation among clonal complexes, variable expression is unlikely to be an issue for vaccine coverage. In particular, regulation of fetA by iron is reduced in many isolates due to a deletion in the sequence bound by the regulatory protein, Fur. Therefore, a vaccine targeting PorA and FetA may provide high levels of protection against meningococcal disease; however, an alternative formulation or immunisation strategy is required to improve coverage against FetA.
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Virulence characterization of Rift Valley fever virus strains and efficacy of glycoprotein subunit vaccines in miceBalogh, Aaron Michael January 1900 (has links)
Master of Science / Department of Diagnostic Medicine/Pathobiology / Juergen A. Richt / Rift Valley fever virus (RVFV) is a vector-borne zoonotic pathogen endemic to sub-Saharan Africa and the Arabian Peninsula that causes severe disease in ruminants and humans. RVFV is a significant threat to US livestock and public health due to a lack of licensed, efficacious vaccines and its ability to become established in non-endemic areas. Subunit vaccine candidates based on RVFV N- and C-terminal glycoproteins (Gn and Gc) are a viable option for use in ruminants due to their ease of production, safety, and ability to induce immune responses that offer differentiation between infected and vaccinated animals (DIVA). Importantly, subunit Gn+Gc vaccine candidates have demonstrated efficacy in sheep. However, despite the efficacy of a dual glycoprotein vaccine, no studies have directly compared protective efficacies of the individual glycoproteins. Furthermore, although RVFV demonstrates 2.1% maximum pairwise amino acid strain divergence within Gn/Gc ectodomains, it remains unclear how this may affect cross-protective vaccine efficacy. In this study, we used a BALB/c mouse model to determine the median lethal dose (LD₅₀) of 3 wildtype RVFV strains and used this information to standardize challenge doses in subsequent vaccine efficacy studies using baculovirus-expressed Gn/Gc antigens derived from RVFV strain Zagazig Hostpital 1977 (ZH548). Strains Kenya 2006 (Ken06) and Saudi Arabia 2001 (SA01) demonstrated equally high virulence (LD₅₀= 7.9pfu), while recombinant strain South Africa 1951 (rSA51) was less virulent (LD₅₀=150pfu). Following prime-boost vaccination, 100% (10/10) of the Gn+Gc vaccinated mice survived challenge with x1000 LD₅₀ Ken06 and SA01, while only 50% (5/10) of Gn+Gc vaccinated mice survived challenge with rSA51. Additionally, 90% (9/10) of Gn-only vaccinated and 40% (4/10) of Gc-only vaccinated mice survived challenge with Ken06. These data suggest that a Gn-only subunit vaccine is an efficacious alternative to dual glycoprotein vaccine candidates and that our ZH548-derived Gn+Gc vaccine has the potential to cross-protect against divergent RVFV strains. Results from this study can be used to optimize current vaccine formulations and inform future vaccine efficacy and licensure studies in ruminants.
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Definition of Bovine Leukocyte Antigen Diversity and Peptide Binding Profiles for Epitope DiscoveryPandya, Mital 01 January 2016 (has links)
The goal of the work presented herein was to further our understanding of Bovine Leukocyte Antigen (BoLA) class I diversity of Holstein cattle and develop tools to measure class I restricted T cell responses to intracellular pathogens such as foot and mouth disease virus (FMDV) following vaccination. BoLA is a highly polymorphic gene region that allows the bovine immune system to differentiate pathogen-infected cells from healthy cells. Immune surveillance by CD8+ T cells plays an important role in clearing viral infections. These CD8+ T cells recognize BoLA class I molecules bearing epitopes (antigenic peptides) of intracellular origin in their peptide binding groove. Polymorphisms in the peptide binding region of class I molecules determine affinity of peptide binding and stability during antigen presentation. Different antigen peptide motifs are associated with specific genetic sequences of class I molecules. In order to better understand the adaptive immune response mediated by BoLA molecules, technologies from human medicine such as high-throughput sequencing, biochemical affinity and stability assays, tetramers and IFN-γ ELIspot assays could be applied. Therefore, it was hypothesized that we can translate these technologies from the study of human T cell responses to the study of cattle immunity.
The first objective was to establish a comprehensive method for genotyping BoLA of Holstein cattle by using Illumina MiSeq, Sanger sequencing and polymerase chain reaction sequence-specific primers (PCR-SSP) (See Chapter 2). This is an important first step in order to study the BoLA restricted immune responses following FMDV vaccination. The second objective was to define the FMDV capsid protein peptide repertoire bound by BoLA class I molecules using bioinformatics and biochemical affinity and stability assays to facilitate the identification of T cell epitopes (See Chapter 3). The third objective was to demonstrate clonal T cell expansion for specific epitope polypeptides using ex-vivo multi-color flow cytometric MHC-epitope complexes (tetramers), followed by IFN-γ production measured by an ELIspot assay to quantify and define the antigen specific response of Holstein cattle to FMDV vaccination (see Chapter 4). In this, my dissertation studies aimed to improve our understanding of the BoLA class I restricted T-cell responses to candidate FMDV vaccines in Holstein cattle. In this manner, my research will improve animal health through the production of assays for characterizing the bovine immune response to intracellular pathogens and enhance vaccine design leading to improved biologicals to protect cattle from devastating infectious diseases.
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Bridging the gap: Using the theory of planned behavior to predict HPV vaccination intentions in menSnipes, Daniel 20 March 2013 (has links)
Genital human papillomavirus (HPV) is the most common sexually transmitted infection (STI) in the US, with an estimated incidence rate of 6.2 million new cases each year. Men have higher instances of certain HPV related outcomes (e.g., head/neck cancers) when compared to women, so male vaccination with the HPV vaccine is also paramount to preventing cancer. The present study examined the theory of planned behavior (TPB) as a model for predicting HPV vaccination intentions among men. Results suggest the TPB was a well-fitting model to the data, but not all aspects of the TPB model were predictive of HPV vaccination intentions. Behavioral beliefs (e.g.., the belief that vaccination will provide a beneficial outcome) were the only significant predictor of HPV vaccination intention in the next 6 months. Perceived norms, motivations to comply with norms, attitudes towards the HPV vaccine, and self-efficacy were not significant predictors of HPV vaccination intentions.
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