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Display of Domain III from Dengue 2 Envelope Protein on HBsAg Virus-like Particles Vectored by Measles Virus

Description

Dengue virus infects millions of people every year. Yet there is still no vaccine available to prevent it. Here we use a neutralizing epitope determinant on the dengue envelope (E) protein as an immunogen to be vectored by a measles

Dengue virus infects millions of people every year. Yet there is still no vaccine available to prevent it. Here we use a neutralizing epitope determinant on the dengue envelope (E) protein as an immunogen to be vectored by a measles virus (MV) vaccine. However the domain III (DIII) of the dengue 2 E protein is too small to be immunogenic by itself. In order for it to be displayed on a larger particle, it was inserted into the amino terminus of small hepatitis B surface antigen (HBsAg, S) coding sequence. To generate the recombinant MV vector and verify the efficiency of this concept, a reverse genetics system was used where the MV vectors express one or two additional transcription units to direct the assembly of hybrid HBsAg particles. Two types of recombinant measles virus were produced: pB(+)MVvac2(DIII-S,S)P and pB(+)MVvac2(DIII-S)N. Virus recovered from pB(+)MVvac2(DIII-S,S)P was viable. An ELISA assay was performed to demonstrate the expression and secretion of HBsAg. Supernatant from MVvac2(DIII-S,S)P infected cells confirmed that hybrid HBsAg-domain III particles with a density similar to traditional HBsAg particles were released. Characteristics of the subviral particle have been analyzed for the successful incorporation of domain III. The replication fitness of the recombinant MV was evaluated using multi-step growth kinetics and showed reduced replication fitness when compared to the parental strain MVvac2. This demonstrates that viral replication is hindered by the addition of the two inserts into MV genome. Further analysis of MVvac2(DIII-S)N is needed to justify immune response studies in a small animal model using both of the generated recombinant vectors.

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2014-05

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Expression of dengue virus envelope glycoproteins using a measles vaccine vector

Description

ABSTRACT In terms of prevalence, human suffering and costs dengue infections are the most important arthropod-borne viral disease worldwide. Dengue virus (DENV) is a mosquito-borne flavivirus and the etiological agent of dengue fever and dengue hemorrhagic fever. Thus, development of

ABSTRACT In terms of prevalence, human suffering and costs dengue infections are the most important arthropod-borne viral disease worldwide. Dengue virus (DENV) is a mosquito-borne flavivirus and the etiological agent of dengue fever and dengue hemorrhagic fever. Thus, development of a safe and efficient vaccine constitutes an urgent necessity. Besides the traditional strategies aim at generating immunization options, the usage of viral vectors to deliver antigenic stimulus in order to elicit protection are particularly attractive for the endeavor of a dengue vaccine. The viral vector (MVvac2) is genetically equivalent to the currently used measles vaccine strain Moraten, which adds practicality to my approach. The goal of the present study was to generate a recombinant measles virus expressing structural antigens from two strains of DENV (DENV2 and DENV4) The recombinant vectors replication profile was comparable to that of the parental strain and expresses either membrane bound or soluble forms of DENV2 and DENV4 E glycoproteins. I discuss future experiments in order to demonstrate its immunogenicity in our measles-susceptible mouse model.

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2013

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Beliefs about change and predicted future health status

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Beliefs about change reflect how we understand phenomena and what kind of predictions we make for the future. Cyclical beliefs about change state that events are in a constant flux, and change is inevitable. Linear beliefs about change state that

Beliefs about change reflect how we understand phenomena and what kind of predictions we make for the future. Cyclical beliefs about change state that events are in a constant flux, and change is inevitable. Linear beliefs about change state that events happen in a non-fluctuating pattern and change is not commonplace. Cultural differences in beliefs about change have been documented across various domains, but research has yet to investigate how these differences may affect health status predictions. The present study addresses this gap by inducing different beliefs about change in a European-American college sample. Health status predictions were measured in terms of predicted likelihood of exposure to the flu virus, of contraction of the flu, and of receiving a flu vaccine. Most differences were observed among those who have a recent history of suffering from the flu. Among them, cyclical thinkers tended to rate their likelihood for exposure and contraction to be higher than linear thinkers. However, linear thinkers indicated that they were more likely to receive a flu vaccine. The different patterns suggest the possibility that cyclical beliefs may activate concepts related to cautionary behaviors or pessimistic biases, while linear beliefs may activate concepts related to taking action and exercising control over the environment. Future studies should examine the interplay between beliefs about change and the nature of the predicted outcome.

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2012

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Biological and immunological characterization of plant-produced HIV-1 Gag/dgp41 virus-like particles

Description

Anti-retroviral drugs and AIDS prevention programs have helped to decrease the rate of new HIV-1 infections in some communities, however, a prophylactic vaccine is still needed to control the epidemic world-wide. Despite over two decades of research, a vaccine against

Anti-retroviral drugs and AIDS prevention programs have helped to decrease the rate of new HIV-1 infections in some communities, however, a prophylactic vaccine is still needed to control the epidemic world-wide. Despite over two decades of research, a vaccine against HIV-1 remains elusive, although recent clinical trials have shown promising results. Recent successes have focused on highly conserved, mucosally-targeted antigens within HIV-1 such as the membrane proximal external region (MPER) of the envelope protein, gp41. MPER has been shown to play critical roles in the viral mucosal transmission, though this peptide is not immunogenic on its own. Gag is a structural protein configuring the enveloped virus particles, and has been suggested to constitute a target of the cellular immunity potentially controlling the viral load. It was hypothesized that HIV-1 enveloped virus-like particles (VLPs) consisting of Gag and a deconstructed form of gp41 comprising the MPER, transmembrane, and cytoplasmic domains (dgp41) could be expressed in plants. Plant-optimized HIV-1 genes were constructed and expressed in Nicotiana benthamiana by stable transformation, or transiently using a tobacco mosaic virus-based expression system or a combination of both. Results of biophysical, biochemical and electron microscopy characterization demonstrated that plant cells could support not only the formation of HIV-1 Gag VLPs, but also the accumulation of VLPs that incorporated dgp41. These particles were purified and utilized in mice immunization experiments. Prime-boost strategies combining systemic and mucosal priming with systemic boosting using two different vaccine candidates (VLPs and CTB-MPR - a fusion of MPER and the B-subunit of cholera toxin) were administered to BALB/c mice. Serum antibody responses against both the Gag and gp41 antigens could be elicited in mice systemically primed with VLPs and these responses could be recalled following systemic boosting with VLPs. In addition, mucosal priming with VLPs allowed for a robust boosting response against Gag and gp41 when boosted with either candidate. Functional assays of these antibodies are in progress to test the antibodies' effectiveness in neutralizing and preventing mucosal transmission of HIV-1. This immunogenicity of plant-based Gag/dgp41 VLPs represents an important milestone on the road towards a broadly-efficacious and inexpensive subunit vaccine against HIV-1.

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Date Created
2011

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Lessons from vaccinia virus post-exposure prophylaxis: insights into control of diseases and epidemics

Description

The concept of vaccination dates back further than Edward Jenner's first vaccine using cowpox pustules to confer immunity against smallpox in 1796. Nevertheless, it was Jenner's success that gave vaccines their name and made vaccinia virus (VACV) of particular interest.

The concept of vaccination dates back further than Edward Jenner's first vaccine using cowpox pustules to confer immunity against smallpox in 1796. Nevertheless, it was Jenner's success that gave vaccines their name and made vaccinia virus (VACV) of particular interest. More than 200 years later there is still the need to understand vaccination from vaccine design to prediction of vaccine efficacy using mathematical models. Post-exposure vaccination with VACV has been suggested to be effective if administered within four days of smallpox exposure although this has not been definitively studied in humans. The first and second chapters analyze post-exposure prophylaxis of VACV in an animal model using v50ΔB13RMγ, a recombinant VACV expressing murine interferon gamma (IFN-γ) also known as type II IFN. While untreated animals infected with wild type VACV die by 10 days post-infection (dpi), animals treated with v50ΔB13RMγ 1 dpi had decreased morbidity and 100% survival. Despite these differences, the viral load was similar in both groups suggesting that v50ΔB13RMγ acts as an immunoregulator rather than as an antiviral. One of the main characteristics of VACV is its resistance to type I IFN, an effect primarily mediated by the E3L protein, which has a Z-DNA binding domain and a double-stranded RNA (dsRNA) binding domain. In the third chapter a VACV that independently expresses both domains of E3L was engineered and compared to wild type in cells in culture. The dual expression virus was unable to replicate in the JC murine cell line where both domains are needed together for replication. Moreover, phosphorylation of the dsRNA dependent protein kinase (PKR) was observed at late times post-infection which indicates that both domains need to be linked together in order to block the IFN response. Because smallpox has already been eradicated, the utility of mathematical modeling as a tool for predicting disease spread and vaccine efficacy was explored in the last chapter using dengue as a disease model. Current modeling approaches were reviewed and the 2000-2001 dengue outbreak in a Peruvian region was analyzed. This last section highlights the importance of interdisciplinary collaboration and how it benefits research on infectious diseases.

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Date Created
2011

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Subunit vaccine to prevent Escherichia coli O157:H7 intestinal attachment and colonization

Description

In the United States, Escherichia coli O157:H7 (E. coli O157:H7) is the most frequent cause of hemolytic uremic syndrome (HUS) and it is also the primary cause of acute renal failure in children. The most common route of the infection

In the United States, Escherichia coli O157:H7 (E. coli O157:H7) is the most frequent cause of hemolytic uremic syndrome (HUS) and it is also the primary cause of acute renal failure in children. The most common route of the infection is ingestion of contaminated meat or dairy product originating from cattle or vegetables contaminated with bovine manure. Since cattle are the main reservoir for human infection with E. coli O157:H7, the reduction of intestinal colonization by these bacteria in cattle is the best approach to prevent human infections. Intimin is an outer membrane protein of E. coli O157:H7 that plays an important role in adhesion of the bacteria to the host cell. Hence, I proposed to express intimin protein in tomato plants to use it as a vaccine candidate to reduce or prevent intestinal colonization of cattle with E. coli O157:H7. I expressed His-tagged intimin protein in tomato plants and tested the purified plant-derived intimin as a vaccine candidate in animal trials. I demonstrated that mice immunized intranasally with purified tomato-derived intimin produced intimin-specific serum IgG1and IgG2a, as well as mucosal IgA. I further demonstrated that mice immunized with intimin significantly reduced time of the E. coli O157:H7 shedding in their feces after the challenge with these bacteria, as compared to unimmunized mice. Shiga toxin is the major virulence factor that contributes to HUS. Since Shiga toxin B subunit has an important role in the attachment of the toxin to its receptor, I fused intimin to Shiga toxin B subunit to create multivalent subunit vaccine and tested the effects upon immunization of mice with the B subunit when combined with intimin. His-tagged intimin, Shiga toxin B subunit, and Shiga toxin-intimin fusion proteins were expressed in E. coli and purified. I demonstrated that this multivalent fusion protein vaccine candidate elicited intimin- and Shiga toxin B-specific IgG1, IgG2a, and IgA antibodies in mice. I also showed a reduction in the duration of the bacterial shedding after the challenge compared to the control sham-immunized groups.

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Date Created
2010

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Use of random peptide reactivities to analyze host immune responses of African swine fever virus infection and immunization

Description

African Swine Fever (ASF), endemic in many African countries, is now spreading to other continents. Though ASF is capable of incurring serious economic losses in affected countries, no vaccine exists to provide immunity to animals. Disease control relies largely on

African Swine Fever (ASF), endemic in many African countries, is now spreading to other continents. Though ASF is capable of incurring serious economic losses in affected countries, no vaccine exists to provide immunity to animals. Disease control relies largely on rapid diagnosis and the implementation of movement restrictions and strict eradication programs. Developing a scalable, accurate and low cost diagnostic for ASF will be of great help for the current situation. CIM's 10K random peptide microarray is a new high-throughput platform that allows systematic investigations of immune responses associated with disease and shows promise as a diagnostic tool. In this study, this new technology was applied to characterize the immune responses of ASF virus (ASFV) infections and immunizations. Six sets of sera from ASFV antigen immunized pigs, 6 sera from infected pigs and 20 sera samples from unexposed pigs were tested and analyzed statistically. Results show that both ASFV antigen immunized pigs and ASFV viral infected pigs can be distinguished from unexposed pigs. Since it appears that immune responses to other viral infections are also distinguishable on this platform, it holds the potential of being useful in developing a new ASF diagnostic. The ability of this platform to identify specific ASFV antibody epitopes was also explored. A subtle motif was found to be shared among a set of peptides displaying the highest reactivity for an antigen specific antibody. However, this motif does not seem to match with any antibody epitopes predicted by a linear antibody epitope prediction.

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Date Created
2011

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Immunogenic subviral particles displaying domain III of dengue 2 envelope protein vectored by measles virus

Description

Vaccines against the arthropod-borne dengue virus (DENV) are still commercially nonexistent. A subunit immunization strategy may be of value, especially if a safe viral vector acts as a biologically active adjuvant. The DENV envelope protein (E), the main target for

Vaccines against the arthropod-borne dengue virus (DENV) are still commercially nonexistent. A subunit immunization strategy may be of value, especially if a safe viral vector acts as a biologically active adjuvant. The DENV envelope protein (E), the main target for neutralizing immune responses, has three conformational domains. The immunoglobulin-like and independently folding domain III (DIII) contains epitopes that elicit highly specific neutralizing antibodies. The hepatitis B small surface antigen (HBsAg, S) was used as a scaffold to display DENV 2 DIII on a virus-like particle (VLP). A measles virus (MV) was engineered to vector HBsAg and the hybrid glycoprotein DIII-HBsAg in two different loci (DIII-S). Despite the relatively deleterious effect on replication caused by the insertion of two transcription cassettes, the recombinant virus MVvac2(DIII-S,S)P induced the secretion of DIII-S hybrid VLP with a similar sucrose density as HBsAg particles (1.10-1.12g/ml) and peaked at 48 h post-infection producing 1.3x106 TCID50/ml infectious MV units in vitro. A second recombinant virus, MVvac2(DIII-S)N, was engineered to vector only the hybrid DIII-S. However, it did not induce the secretion of hybrid HBsAg particles in the supernatant of infected cells. The immunogenicity of the recombinant viruses was tested in a MV-susceptible small animal model, the experimental group which received two 105 TCID50 I.P. doses of MVvac2(DIII-S,S)P in a 28 day interval developed a robust immune response against MV (1:1280), HBsAg (787 mIU/ml) and DENV2 (Log10 neutralization index of 1.2) on average. In summary, it is possible to display DENV E DIII on hybrid HBsAg particles vectored by MV that elicit an immune response. This forms the basis for a potential vaccine platform against DENV.

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Date Created
2015

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Searching for an HIV vaccine: a heterologous prime-boost system using replicating vaccinia virus and plant-produced virus-like particles

Description

The HIV-1 pandemic continues to cause millions of new infections and AIDS-related deaths each year, and a majority of these occur in regions of the world with limited access to antiretroviral therapy. Therefore, an HIV-1 vaccine is still desperately needed.

The HIV-1 pandemic continues to cause millions of new infections and AIDS-related deaths each year, and a majority of these occur in regions of the world with limited access to antiretroviral therapy. Therefore, an HIV-1 vaccine is still desperately needed. The most successful HIV-1 clinical trial to date used a non-replicating canarypox viral vector and protein boosting, yet its modest efficacy left room for improvement. Efforts to derive novel vectors which can be both safe and immunogenic, have spawned a new era of live, viral vectors. One such vaccinia virus vector, NYVAC-KC, was specifically designed to replicate in humans and had several immune modulators deleted to improve immunogenicity and reduce pathogenicity. Two NYVAC-KC vectors were generated: one expressing the Gag capsid, and one with deconstructed-gp41 (dgp41), which contains an important neutralizing antibody target, the membrane proximal external region (MPER). These vectors were combined with HIV-1 Gag/dgp41 virus-like particles (VLPs) produced in the tobacco-relative Nicotiana benthamiana. Different plant expression vectors were compared in an effort to improve yield. A Geminivirus-based vector was shown to increase the amount of MPER present in VLPs, thus potentially enhancing immunogenicity. Furthermore, these VLPs were shown to interact with the innate immune system through Toll-like receptor (TLR) signaling, which activated antigen presenting cells to induce a Th2-biased response in a TLR-dependent manner. Furthermore, expression of Gag and dgp41 in NYVAC-KC vectors resulted in activation of antiviral signaling pathways reliant on TBK1/IRF3, which necessitated the use of higher doses in mice to match the immunogenicity of wild-type viral vectors. VLPs and NYVAC-KC vectors were tested in mice, ultimately showing that the best antibody and Gag-specific T cell responses were generated when both components were administered simultaneously. Thus, plant-produced VLPs and poxvirus vectors represent a highly immunogenic HIV-1 vaccine candidate that warrants further study.

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Date Created
2016

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Analysis of small molecule interactions in biological systems: the study of potential treatments for addiction and disease

Description

The ability to manipulate the interaction between small molecules and biological macromolecules towards the study of disease pathogenesis has become a very important part of research towards treatment options for various diseases. The work described here shows both the use

The ability to manipulate the interaction between small molecules and biological macromolecules towards the study of disease pathogenesis has become a very important part of research towards treatment options for various diseases. The work described here shows both the use of DNA oligonucleotides as carriers for a nicotine hapten small molecule, and the use of microsomes to study the stability of compounds derived to treat mitochondrial diseases.

Nicotine addiction is a worldwide epidemic because nicotine is one of the most widely used addictive substances. It is linked to early death, typically in the form of heart or lung disease. A new vaccine conjugate against nicotine held within a DNA tetrahedron delivery system has been studied. For this purpose, several strands of DNA, conjugated with a modified dTpT having three or six carbon atom alkynyl linkers, have been synthesized. These strands have later been conjugated to three separate hapten small molecules to analyze which conjugates formed would be optimal for further testing in vivo.

Mitochondrial diseases are hard to treat, given that there are so many different variations to treat. There is no one compound that can treat all mitochondrial and neurodegenerative diseases; however, improvements can be made to compounds currently under study to improve the conditions of those afflicted. A significant issue leading to compounds failing in clinical trials is insufficient metabolic stability. Many compounds have good biological activity, but once introduced to an animal, are not stable enough to have any effect. Here, several synthesized compounds have been evaluated for metabolic stability, and several showed improved stability, while maintaining biological activity.

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Date Created
2016