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The Development of a Plant-Expressed M2e-Based Universal Influenza Vaccine

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Influenza is a deadly disease for which effective vaccines are sorely lacking. This is largely due to the phenomena of antigenic shift and drift in the influenza virus's surface proteins, hemagglutinin (HA) and neuraminidase (NA). The ectodomain of the matrix

Influenza is a deadly disease for which effective vaccines are sorely lacking. This is largely due to the phenomena of antigenic shift and drift in the influenza virus's surface proteins, hemagglutinin (HA) and neuraminidase (NA). The ectodomain of the matrix 2 protein (M2e) of influenza A, however, has demonstrated high levels of conservation. On its own it is poorly immunogenic and offers little protection against influenza infections, but by combining it with a potent adjuvant, this limitation may be overcome. Recombinant immune complexes, or antigens fused to antibodies that have been engineered to form incredibly immunogenic complexes with one another, were previously shown to be useful, immunogenic platforms for the presentation of various antigens and could provide the boost in immunogenicity that M2e needs to become a powerful universal influenza A vaccine. In this thesis, genetic constructs containing geminiviral replication proteins and coding for a consensus sequence of dimeric M2e fused to antibodies featuring complimentary epitopes and epitope tags were generated and used to transform Agrobacterium tumefaciens. The transformed bacteria was then used to cause Nicotiana benthamiana to transiently express M2e-RICs at very high levels, with enough RICs being gathered to evaluate their potency in future mouse trials. Future directions and areas for further research are discussed.

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2018-05

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Characterization of Zika Virus Inactivated by Ultrashort Pulsed Laser as a Source for Vaccine Development

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The objective of this thesis was to determine whether Zika Virus (ZIKV) can be effectively inactivated by Selective Photonic Disinfection (SEPHODIS) and determine whether key proteins involved in the infection process are preserved, making SEPHODIS a possible source for vaccine

The objective of this thesis was to determine whether Zika Virus (ZIKV) can be effectively inactivated by Selective Photonic Disinfection (SEPHODIS) and determine whether key proteins involved in the infection process are preserved, making SEPHODIS a possible source for vaccine development. As of January 2018, there have been 3,720 confirmed cases of Congenital Zika Syndrome in infants, making a Zika Vaccine a high priority (Mitchell, 2018). SEPHODIS is a process that involves prolonged exposure of an object to a pulsing laser which can render it ineffective. Initially, ZIKV was subjected to laser inactivation for 6 hours, then a plaque assay was performed on both laser-treated and control samples. ZIKV was inactivated two-fold? after laser treatment, when compared with control, as indicated by the plaque assay results. Additionally, both samples were submitted to ELISA to evaluate antigenicity with a panel of monoclonal and human sera. As a second control, virus inactivated by formaldehyde (2%) was used. ELISA results showed that antigenicity of some proteins were preserved while others were probably disturbed. However, ELISA results show that ZIKV envelope protein (E-protein), the protein responsible for viral entry into cells, was effectively preserved after laser-treatment, implying that if laser parameters were tweaked to obtain more complete inactivation, then SEPHODIS may be an appropriate source for the development of a vaccine.

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2018-05

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Detoxifying Lipid A in Agrobacterium tumefaciens

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Agrobacterium tumefaciens has the ability to transfer its tumor inducing (Ti) plasmid into plant cells. In the last decade, agroinfiltration of Nicotiana benthamiana plants has shown promising results for recombinant protein production. However, A. tumefaciens produce endotoxins in the form

Agrobacterium tumefaciens has the ability to transfer its tumor inducing (Ti) plasmid into plant cells. In the last decade, agroinfiltration of Nicotiana benthamiana plants has shown promising results for recombinant protein production. However, A. tumefaciens produce endotoxins in the form of lipopolysaccharides (LPS), a component of their outer membrane that can induce organ failure and septic shock. Therefore, we aimed to detoxify A. tumefaciens by modifying their Lipid A structure, the toxic region of LPS, via mutating the genes for lipid A biosynthesis. Two mutant strains of A. tumefaciens were infiltrated into N. benthamiana stems to test for tumor formation to ensure that the detoxifying process did not compromise the ability of gene transfer. Our results demonstrated that A. tumefaciens with both single and double mutations retained the ability to form tumors. Thus, these mutants can be utilized to generate engineered A. tumefaciens strains for the production of plant-based pharmaceuticals with low endotoxicity.

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2018-05

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Effects of LCMV Infection on Murine Fetal Development in Immunized Mothers

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Despite a continuously growing body of evidence that they are one of the major causes of pregnancy loss, preterm birth, pregnancy complications, and developmental abnormalities leading to high rates of morbidity and mortality, viruses are often overlooked and underestimated as

Despite a continuously growing body of evidence that they are one of the major causes of pregnancy loss, preterm birth, pregnancy complications, and developmental abnormalities leading to high rates of morbidity and mortality, viruses are often overlooked and underestimated as teratogens. The Zika virus epidemic beginning in Brazil in 2015 brought teratogenic viruses into the spotlight for the public health community and popular media, and its infamy may bring about positive motivation and funding for novel treatments and vaccination strategies against it and a variety of other viruses that can lead to severe congenital disease. Lymphocytic choriomeningitis virus (LCMV) is famous in the biomedical community for its historic and continued utility in mouse models of the human immune system, but it is rarely a source of clinical concern in terms of its teratogenic risk to humans, despite its ability to cause consistently severe ocular and neurological abnormalities in cases of congenital infection. Possibilities for a safe and effective LCMV vaccine remain difficult, as the robust immune response typical to LCMV can be either efficiently protective or lethally pathological based on relatively small changes in the host type, viral strain, viral dose, method of infection/immunization, or molecular characteristics of synthetic vaccination. Introducing the immunologically unique state of pregnancy and fetal development to the mix adds complexity to the process. This thesis consists of a literature review of teratogenic viruses as a whole, of LCMV and its complications during pregnancy, of LCMV immunopathology, and of current understanding of vaccination against LCMV and against other teratogenic viruses, as well as a hypothetical experimental design intended to initially bridge the gaps between LCMV vaccinology and LCMV teratogenicity by bringing a vaccine study of LCMV into the context of viral challenge during pregnancy.

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2020-05

Panic and Potential Pandemia

Description

The United States has been facing a resurgence of vaccine preventable infectious diseases. Non-medical vaccination exemptions (NMEs) which include religious exemptions and philosophical vaccine exemptions are contributing factors in state vaccination rates dropping. The policies surrounding such exemptions vary from

The United States has been facing a resurgence of vaccine preventable infectious diseases. Non-medical vaccination exemptions (NMEs) which include religious exemptions and philosophical vaccine exemptions are contributing factors in state vaccination rates dropping. The policies surrounding such exemptions vary from state to state. Some states with higher rates of nonmedical vaccine exemptions are dealing with repercussions for this including vaccination rates falling below desired herd immunity and thus putting vulnerable populations such as those who are immunocompromised, too young for vaccination and the elderly at a higher risk.

This thesis aims to examine vaccine preventable re-emerging infectious diseases in the United States with the objective of reaching vaccine hesitant populations and providing them with the tools to make informed decisions to seek out immunizations. This will be done by exploring five different diseases and infections, discussing why some individuals feel hesitant to get immunizations, examining how nonmedical vaccine exemptions are correlated to increased cases of disease outbreaks, looking into state laws specifically focused on countering nonmedical vaccine exemptions and the steps that can be taken moving forward.

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2020-05

Point-of-Care Testing for Detection of Measles and Mumps Immunity

Description

Measles and mumps are highly contagious, vaccine-preventable diseases with cases continuing to persist in high two-dose vaccinated populations. Recent outbreaks on university and college campuses across the United States prompt a need for further understanding of the immunity levels afforded

Measles and mumps are highly contagious, vaccine-preventable diseases with cases continuing to persist in high two-dose vaccinated populations. Recent outbreaks on university and college campuses across the United States prompt a need for further understanding of the immunity levels afforded by the MMR vaccine which has significantly decreased incidence rates of measles and mumps since it was introduced.
Current methods for IgG antibody detection include enzyme immunoassays (EIA) such as the commercially available Diamedix Immunosimplicity® Measles IgG test kit and the Diamedix Immunosimplicity® Mumps IgG test kit. EIAs generally provide high sensitivity and strong specificity, however, there is a need for rapid screening of measles and mumps specific immunity in outbreak and resource-limited areas which could be solved by use a point-of-care (POC) platform.
This study aims to optimize a point-of-care device for the multiplexed detection of MeV, MuV, and RuV IgG antibodies in sera and to compare the sensitivity to commercial enzyme immunoassays. The IgG antibody levels to MeV and MuV were measured using EIA test kits for a total of 44 healthy serum samples. Of the samples, 6% were seronegative for MeV-specific IgG antibodies and 75% were seronegative for MuV-specific antibodies, showing low correlation of IgG antibody levels between both viruses.
To improve the sensitivity of the POC device, multiple conjugated fluorescent secondary antibodies were tested with different surface chemistries. Signal detection was measured using the pre-developed four-site slide reader. Preliminary data show that Nile Red microspheres provide robust signal detection and should be the secondary antibody of choice when sera are tested for IgG antibodies using the POC platform in future work.

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2017-05

Developing and Pilot Testing Digital Storytelling Interventions to Promote HPV Vaccinations among Vietnamese American Adolescents

Description

Significance Background: Human papilloma virus (HPV) is the most common sexually transmitted infection, affecting 79 million Americans today and an additional 14 million Americans becoming infected with HPV each year. HPV infection may lead to the development of genital warts

Significance Background: Human papilloma virus (HPV) is the most common sexually transmitted infection, affecting 79 million Americans today and an additional 14 million Americans becoming infected with HPV each year. HPV infection may lead to the development of genital warts and several types of cancers including both cervical and oropharyngeal cancers. The promotion of currently available HPV vaccines is important to prevent HPV transmission and reduce the prevalence of the comorbidities associated with infection. Promotion to Vietnamese-Americans in particular is important because of the increased rates of cervical cancers seen in this population. As Vietnamese-American mothers often act as the primary healthcare decision maker for their children, they were chosen as the target population for this intervention. Purpose: This study aims to (1) develop personal digital stories about HPV and HPV vaccination among Vietnamese women with adolescent children who are vaccinated against HPV; and (2) share these stories with a group of Vietnamese American mothers and assess the effect of the stories in changing the attitudes, beliefs, and intention to vaccinate for HPV. Methods: This study used a two-step process to design, implement, and evaluate digital stories to improve Vietnamese mothers' attitudes, beliefs, and intention to vaccinate their adolescent children against HPV. The first step was a formative research design to develop the digital stories. The second step was quasi-experimental with a pre and posttest design to evaluate the effect of the stories. Results: The first phase has produced two digital stories which will be screened recruitment has been completed for phase two. Content analysis showed the importance of community resources, the desire to protect children, a history of familial and/or personal cancer, concerns about side effects, and the influence of healthcare providers as themes in both stories. Recruitment efforts are underway to recruit eligible Vietnamese mothers to assess the effect of these stories. Data collection is ongoing. Conclusions and lessons learned: The project has yielded two digital stories and recruitment for phase two is underway. This project has been successful in obtaining IRB approval, recruiting phase one participants, holding a digital storytelling workshop, designing the phase two survey, and beginning data collection efforts. The phase two recruitment has been challenging and will necessitate a change in strategy to find participants.

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2017-05

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Perceptions of Vaccine Risks and Effectiveness Among ASU Students

Description

The development of safe and effective vaccines has been one of the greatest public achievements of the 20th century. However, there is still considerable public debate about the relative health costs and benefits of vaccines, and the information and misinformation

The development of safe and effective vaccines has been one of the greatest public achievements of the 20th century. However, there is still considerable public debate about the relative health costs and benefits of vaccines, and the information and misinformation spread through these debates can have a direct impact on vaccination and whether or not herd immunity will continue in the United States for different diseases. To understand perceptions of vaccine risks and effectiveness among young adults in the U.S., this study describes Arizona State University students' perceptions of the harms and benefits of vaccines. A preliminary free list (n=30) identified what vaccines ASU college students were most likely to recall spontaneously. The six vaccines most commonly mentioned by ASU students were: influenza (flu), chickenpox, HPV, polio, MMR, and smallpox. Using these top six vaccines, we then developed a second survey about the knowledge and perceptions of each of these vaccines and vaccines as a whole. We found that students generally perceived vaccines as safe and important to their health, but they maintained an overall lack of understanding of how vaccines work and what they protect against. While this study is only a preliminary investigation into the perceptions of ASU college students on six commonly mentioned vaccines, this could lead to investigations on how to educate and promote the usage of vaccines to college students.

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Date Created
2017-12

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Linking Immunologic and Epidemiologic Models of Virus Transmission and Susceptibility

Description

Memory CD8+ T-cells can persist in the absence of antigen, primed for immediate activation and proliferation if later exposed to the same antigen. These cytotoxic lymphocytes provide long-term immunity following an acute infection. Studies have observed that intermediate levels of

Memory CD8+ T-cells can persist in the absence of antigen, primed for immediate activation and proliferation if later exposed to the same antigen. These cytotoxic lymphocytes provide long-term immunity following an acute infection. Studies have observed that intermediate levels of general T cell transfer prior to infection may cause an inappropriate response resulting in increased pathology rather than prevention. Therefore, our study focused on a memory CD8 T-cell therapy using lymphocytic choriomeningitis virus (LCMV) specific splenocytes, which activate and proliferate at an accelerated pace compared to that of naive T-cells. LCMV is a natural murine pathogen which also poses a zoonotic infection threat to humans, and the effect of immune cell vaccination therapies for LCMV is not fully understood. We observed the effect of multiple memory CD8 T cell dosage levels on overall disease and memory CD8 T-cell response to the virus. Infection by exposure to a carrier was shown to have a reduced impact on mice receiving higher doses of memory T cells prior to infection compared to mice receiving less or no memory cells. Higher presence of activated memory cells were shown to correlate with less disease-related weight loss and accelerated recovery times. Survival rate after exposure to carriers was not shown to be affected by dosage level, warranting further research regarding the prevalence of the immunopathology observed in other studies in natural murine transmission models.

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2016-05

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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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Date Created
2014-05