Matching Items (9)
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- All Subjects: Vaccines
- All Subjects: Oncolytic Virotherapy
- Creators: McFadden, Grant
- Creators: Reyes del Valle, Jorge
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 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.
ContributorsAbdelgalel, Rowida (Author) / Reyes del Valle, Jorge (Thesis advisor) / Hogue, Brenda (Committee member) / Frasch, Wayne D (Committee member) / Arizona State University (Publisher)
Created2013
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 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.
ContributorsHarahap, Indira Saridewi (Author) / Reyes del Valle, Jorge (Thesis director) / Hogue, Brenda (Committee member) / Misra, Rajeev (Committee member) / Barrett, The Honors College (Contributor) / T. Denny Sanford School of Social and Family Dynamics (Contributor) / School of Human Evolution and Social Change (Contributor) / School of Life Sciences (Contributor)
Created2014-05
Description
In this study, we investigated the inactivation of wild-type vMyx-GFP (MYXV) using different methods. Assays were performed in vitro to test the following inactivation methods: heat, longwave UV only, longwave UV with psoralen (P + LWUV), and psoralen (P) only. In vitro assays demonstrated that the psoralen alone treatment did not cause any inactivation. These results showed that effective inactivation using psoralen was likely reliant on subsequent UV irradiation, creating a synergistic effect. Additionally, the UV and P + LWUV treatment demonstrated inactivation of MYXV, although by different mechanisms, as the UV-only treated virus demonstrated background infection, while P + LWUV treated virus did not. In mice, P + LWUV and UV treatment of MYXV demonstrated to be effective inactivation methods and likely preserved the antigenic epitopes of MYXV, allowing for the production of neutralizing antibodies in mice. More research is recommended on the heat treatment of MYXV as neutralizing antibodies were not observed, possibly due to the treatment denaturing antigenic epitopes or needing more booster injections to reach the threshold antibody concentration for protection. Furthermore, we demonstrated that the intraperitoneal (IP) injection of inactivated MYXV was superior to the subcutaneous injection in eliciting a strong immune response. The increased neutralizing antibodies observed after IP injection could be due to the advantage that the IP route has of reaching lymphoid tissue faster.
ContributorsSprout, Jamie (Co-author) / Davoudi, Sahar (Co-author) / McFadden, Grant (Thesis director) / Rahman, Masmudur (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
Description
With the advent of precision medicine, oncologists aim to target tumors that do not respond well to conventional treatment. One such therapy is oncolytic virotherapy, a treatment reliant on viral replication for tumor specific killing. Downregulation of the proteins RIP3 kinase, DAI or MLKL can result in a nonfunctional programmed necroptotic cell death pathway, common amongst breast cancer and melanoma. Vaccinia virus (VACV) mutants with a nonfunctional E3 protein are able to selectively replicate in necroptosis deficient cells but not in necroptosis competent cells, making them potential candidates for oncolytic virotherapy. In order to establish the efficacy and selectivity of this treatment, an accurate tumor model is required. Eight established breast adenocarcinomas and two established melanomas were selected as potential candidates, both human and murine. A pan screening method for necroptosis was established utilizing western blot analysis for expression of aforementioned proteins following various induction methods such as IFN α or VACV infection. In addition, live cell imaging after treatment with tumor necrosis factor (TNFα) and the pan-caspase inhibitor zVAD-fmk was used as a method to visualize necroptosis pathway functionality. Based on these results, cell lines will be selected and modified to create a breast cancer model with cells that are syngeneic, differing only in expression of either RIP3. VACV can be tested for tumor volume reduction in these models to ask if RIP3 expression affects efficacy of mutant VACV as an oncolytic virus.
ContributorsKumar, Aradhana (Author) / Jacobs, Bertram (Thesis director) / McFadden, Grant (Committee member) / Borad, Mitesh (Committee member) / School of Life Sciences (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Novel biological strategies for cancer therapy have recently been able to generate antitumor effects in the clinic. Of these new advancements, oncolytic virotherapy seems to be a promising strategy through a dual mechanism of oncolysis and immunogenicity of the host to the target cells. Myxoma virus (MYXV) is an oncolytic poxvirus that has a natural tropism for European rabbits, being nonpathogenic in humans and all other known vertebrates. MYXV is able to infect cancer cells which, due to mutations, have defects in many signaling pathways, notably pathways involved in antiviral responses. While MYXV alone elicits lysis of cancer cells, recombinant techniques allow for the implementation of transgenes, which have the potential of ‘arming’ the virus to enhance its potential as an oncolytic virus. The implementation of certain transgenes allow for the promotion of robust anti-tumor immune responses. To investigate the potential of immune-inducing transgenes in MYXV, in vitro experiments were performed with several armed recombinant MYXVs as well as unarmed wild-type and rabbit-attenuated MYXV. As recent studies have shown the ability of MYXV to uniquely target malignant human hematopoietic stem cells, the potential of oncolytic MYXV armed with immune-inducing transgenes was investigated through in vitro killing analysis using human acute myeloid leukemia (AML) and multiple myeloma (MM) cell lines. Furthermore, in vitro experiments were also performed using primary bone marrow (BM) cells obtained from human patients diagnosed with MM. In this study, armed MYXV-infected human AML and MM cells resulted in increased cell death relative to unarmed MYXV-infected cells, suggesting enhanced killing via induced mechanisms of cell death from the immune-inducing transgenes. Furthermore, increased killing of primary BM cells with multiple myeloma was seen in armed MYXV-infected primary cells relative to unarmed MYXV-infected primary cells.
ContributorsMamola, Joseph (Author) / McFadden, Grant (Thesis director) / Jacobs, Bertram (Committee member) / Blattman, Joseph (Committee member) / School of Life Sciences (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Oncolytic virotherapy (OV) is the use of viruses that do not target normal human cells to infect and destroy cancer cells; some also stimulate the immune system against the tumors. Myxoma virus (VMYX) is a candidate for use as an oncolytic agent, as it is not pathogenic to humans and can infect a variety of human cancer cells. VMYX also can initiate immune responses against the virus-infected tumor. Thus, we investigated the oncolytic efficacy of a few recombinant constructs of VMYX on triple-negative breast cancer (TNBC), a highly aggressive subtype of breast cancer with limited treatment options. TNBC lacks an estrogen receptor, progesterone receptor, and HER2, which render hormone-based therapies useless. Further challenges of TNBC include early metastasis and recurrence, as well as poor prognosis due to a lack of molecular targets. Here, we utilized 4T1-Luc2 cells, an in vitro mouse model of TNBC, to examine the oncolytic potential of recombinant viral constructs of VMYX. Ability to infect was measured by fluorescence intensity, while ability to induce cytotoxicity was measured through MTS and SYTOX assays. Further characterization of cell death was performed using Caspase 3/7 activity assay, immunofluorescent staining and confocal microscopy to detect ecto-expression of calreticulin, and ATP release assays. We demonstrated the ability of recombinant VMYX constructs to infect and induce cell death in 4T1-Luc2 cells. VMYX-p14-FAST-GFP induced the most cell death, while VMYX-M011LKO-GFP best activated Caspase 3/7. Through ATP release assays and examination of ecto-expression of calreticulin, preliminary data indicated that VYX-135KO-GFP is unable to stimulate immunogenic cell death, a form of cell death that stimulates an adaptive immune response, in these cells. Future directions include further characterization of cell death in vitro, as well as in vivo studies.
ContributorsBelmont, Laura (Author) / McFadden, Grant (Thesis director) / Chen, Julian (Committee member) / School of Life Sciences (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Lung metastatic cancers represent a major challenge in both basic and clinical cancer research. The ability to treat lung metastases to date has been challenging, current treatment paradigms are a mix of classic radiotherapy, chemotherapies and tumor-targeted therapies, with no one treatment that is effective for all tumors. Oncolytic viruses (OVs) represent a new therapeutic modality for hard-to-treat tumors. However, major questions still exist in the field, especially around how to therapeutically arm and deliver OVs to sites of disseminated tumors. To address this need, oncolytic myxoma viruses (MYXV) that expresses TNF superfamily member transgenes (vMYX-hTNF or vMyx-mLIGHT) were tested in an immunocompetent syngeneic lung metastatic murine osteosarcoma model. Three versions of this model were used; 1-an early intervention model, 2-an established tumor model, defined by both average tumor burden and failure of anti-PD-L1 and vMyx-TNF monotherapies, and 3-a late-stage disease model, defined by the failure the combination of vMyx-hTNF/PBMCs and anti-PD-L1 therapy. These three models were designed to test different questions about therapeutic efficacy of armed MYXV and delivery of MYXV to lung metastases. In the early intervention model, vMyx-hTNF was found to be an effective therapy, especially when delivered by leukocyte carrier cells (either bone marrow or PBMCs). Next, the combination of immune checkpoint inhibitors, including anti-PD-L1, anti-PD-1 and anti-CTLA-4, with vMyx-TNF/PBMCs were found to increase efficacy in treated mice compared to monotherapies. The established model was used to test potential synergy of vMyx-hTNF with anti-PD-L1 therapy. This model was defined by the failure of the monotherapies, however, in combination, treated mice survived significantly longer, and had lower average tumor burden throughout. This model was also used to test tumor specific delivery using ex vivo loaded PBMCs as carrier cells. Using MYXV expressing Tdtomato, PBMCs were found to deliver MYXV to tumors more effectively than free virus. In the most stringent late-stage disease model, vMyx-mLIGHT/PBMCs and vMyx-mLIGHT/PBMCs plus anti-PD-1 were tested and found to be efficacious where combination vMyx-TNF/PBMCs plus PD-1 failed. These results taken together show that TNFSF arming of MYXV, especially when delivered by autologous PBMCs, represents a new potential treatment strategy for lung metastatic tumors.
ContributorsChristie, John Douglas (Author) / McFadden, Grant (Thesis advisor) / Blattman, Joseph (Committee member) / Jacobs, Bertram (Committee member) / Anderson, Karen (Committee member) / Arizona State University (Publisher)
Created2021
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 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.
ContributorsHarahap, Indira (Author) / Reyes del Valle, Jorge (Thesis advisor) / Hogue, Brenda G (Thesis advisor) / Lake, Douglas (Committee member) / Mason, Hugh (Committee member) / Arizona State University (Publisher)
Created2015
Description
Despite the approval of a Dengue virus (DV) vaccine in five endemic countries, dengue prevention would benefit from an immunization strategy highly immunogenic in young infants and not curtailed by viral interference. Problematically, infants younger than 9 year of age, whom are particularly prone to Dengue severe infection and death, cannot be immunized using current approved DV vaccine. The most important issues documented so far are the lack of efficiency and enhancement of the disease in young seronegative recipients, as well as uneven protection against the four DV serotypes. Based on data from clinical trials that showed enhanced performance of dengue vaccines when the host has previous anti-flaviviral immunity, I proposed here an attractive solution to complement the current vaccine: a recombinant measles vaccine vectoring dengue protective antigens to be administered to young infants. I hypothesized that recombinant measles virus expressing Dengue 2 and 4 antigens would successfully induce neutralizing responses against DV2 and 4 and the vaccine cocktail of this recombinant measles can prime anti-flaviviral neutralizing immunity. For this dissertation, I generated and performed preclinical immune assessment for four novel Measles-Dengue (MV-DV) vaccine candidates. I generated four MVs expressing the pre membrane (prM) and full length or truncated (90%) forms of the major envelope (E) from DV2 and DV4. Two virus, MVvac2-DV2(prME)N and MVvac2-DV4(prME), expressed high levels of membrane associated full-length E, while the other two viruses, MVvac2-DV2(prMEsol)N and MVvac2-DV4(prMEsol)N, expressed and secreted truncated, soluble E protein to its extracellular environment. The last two vectored vaccines proved superior anti-dengue neutralizing responses comparing to its corresponding full length vectors. Remarkably, when MVvac2-DV2/4(prMEsol)N recombinant vaccines were combined, the vaccine cocktail was able to prime cross-neutralizing responses against DV 1 and the relatively distant 17D yellow fever virus attenuated strain. Thus, I identify a promising DV vaccination strategy, MVvac2-DV2/4(prMEsol)N, which can prime broad neutralizing immune responses by using only two of the four available DV serotypes. The current MV immunization scheme can be advantageus to prime broad anti-flaviviral neutralizing immunity status, which will be majorly boosted by subsequent chimeric Dengue vaccine approaches.
ContributorsAbdelgalel, Rowida (Author) / Reyes del Valle, Jorge (Thesis advisor) / Mason, Hugh (Thesis advisor) / Lake, Douglas (Committee member) / Stout, Valerie (Committee member) / Frasch, Wayne (Committee member) / Arizona State University (Publisher)
Created2016