ASU Scholarship Showcase

Non-typhoidal Salmonella are associated with gastrointestinal disease worldwide and invasive disease in Africa. We constructed novel bivalent vaccines through the recombinant expression of heterologous O-antigens from Salmonella Choleraesuis in Salmonella Typhimurium. A recombinant Asd⁺ plasmid pCZ1 with the cloned Salmonella Choleraesuis O-antigen gene cluster was introduced into three constructed Salmonella Typhimurium Δasd mutants: SLT11 (ΔrfbP), SLT12 (ΔrmlB-rfbP) and SLT16 (ΔrfbP ∆pagL::TT araCP_BADrfbP). Immunoblotting demonstrated that SLT11 (pCZ1) and SLT12 (pCZ1) efficiently expressed the heterologous O-antigen. In the presence of arabinose, SLT16 (pCZ1) expressed both the homologous and heterologous O-antigens, whereas in the absence of arabinose, SLT16 (pCZ1) mainly expressed the heterologous O-antigen. We deleted the crp/cya genes in SLT12 (pCZ1) and SLT16 (pCZ1) for attenuation purposes, generating the recombinant vaccine strains SLT17 (pCZ1) and SLT18 (pCZ1). Immunization with either SLT17 (pCZ1) or SLT18 (pCZ1) induced specific IgG against the heterologous O-antigen, which mediated significant killing of Salmonella Choleraesuis and provided full protection against a lethal homologous challenge in mice. Furthermore, SLT17 (pCZ1) or SLT18 (pCZ1) immunization resulted in 83% or 50% heterologous protection against Salmonella Choleraesuis challenge, respectively. Our study demonstrates that heterologous O-antigen expression is a promising strategy for the development of multivalent Salmonella vaccines.

Astronauts are exposed to a unique combination of stressors during spaceflight, which leads to alterations in their physiology and potentially increases their susceptibility to disease, including infectious diseases. To evaluate the potential impact of the spaceflight environment on the regulation of molecular pathways mediating cellular stress responses, we performed a first-of-its-kind pilot study to assess spaceflight-related gene-expression changes in the whole blood of astronauts. Using an array comprised of 234 well-characterized stress-response genes, we profiled transcriptomic changes in six astronauts (four men and two women) from blood preserved before and immediately following the spaceflight. Differentially regulated transcripts included those important for DNA repair, oxidative stress, and protein folding/degradation, including HSP90AB1, HSP27, GPX1, XRCC1, BAG-1, HHR23A, FAP48, and C-FOS. No gender-specific differences or relationship to number of missions flown was observed. This study provides a first assessment of transcriptomic changes occurring in the whole blood of astronauts in response to spaceflight.

Narita 104 virus is a human pathogen belonging to the norovirus (family Caliciviridae) genogroup II. Noroviruses cause epidemic gastroenteritis worldwide. To explore the potential of developing a plant-based vaccine, a plant optimized gene encoding Narita 104 virus capsid protein (NaVCP) was expressed transiently in Nicotiana benthamiana using a tobacco mosaic virus expression system. NaVCP accumulated up to approximately 0.3 mg/g fresh weight of leaf at 4 days postinfection. Initiation of hypersensitive response-like symptoms followed by tissue necrosis necessitated a brief infection time and was a significant factor limiting expression. Transmission electron microscopy of plant-derived NaVCP confirmed the presence of fully assembled virus-like particles (VLPs). In this study, an optimized method to express and partially purify NaVCP is described. Further, partially purified NaVCP was used to immunize mice by intranasal delivery and generated significant mucosal and serum antibody responses. Thus, plant-derived Narita 104 VLPs have potential for use as a candidate subunit vaccine or as a component of a multivalent subunit vaccine, along with other genotype-specific plant-derived VLPs.

Human herpesvirus 8 (HHV8) infection leads to potent activation of nuclear factor kappa B (NFκB) in primary and transformed cells. We used recombinant HHV8 (rKSHV.219) expressing green fluorescent protein under the constitutive cellular promoter elongation factor 2α and red fluorescent protein under an early HHV8 lytic gene promoter T1.1 to monitor replication during infection of human foreskin fibroblasts (HF), noting changes in NFκB activity. In primary HF, NFκB levels do not affect the ability of HHV8 to establish infection or maintain latency. Furthermore, there was no effect on the percent of cells undergoing reactivation from latency, and there were similar numbers of released and cell-associated HHV8 viral particles following reactivation in the presence of inhibitors. Reactivation of HHV8 in latently infected HF in the presence of NFκB inhibitors resulted in production of viral particles that did not efficiently establish infection, due to deficiencies in binding and/or entry into normally permissive cells. Exogenous expression of glycoprotein M, an envelope protein involved in viral binding and entry, was able to partially overcome the deficiency induced by NFκB inhibitors. Our data indicate that in primary cells, NFκB is not required for infection, establishment of latency, or entry into the lytic cycle, but is required for the expression of virion associated genes involved in the initial steps of virion infectivity. These studies suggest that strategies to inhibit NFκB may prevent HHV8 spread and should be considered as a potential therapeutic target for preventing HHV8 associated diseases.

We described the rapid production of the domain III (DIII) of the envelope (E) protein in plants as a vaccine candidate for West Nile Virus (WNV). Using various combinations of vector modules of a deconstructed viral vector expression system, DIII was produced in three subcellular compartments in leaves of Nicotiana benthamiana by transient expression. DIII expressed at much higher levels when targeted to the endoplasmic reticulum (ER) than that targeted to the chloroplast or the cytosol, with accumulation level up to 73 μg DIII per gram of leaf fresh weight within 4 days after infiltration. Plant ER-derived DIII was soluble and readily purified to > 95% homogeneity without the time-consuming process of denaturing and refolding. Further analysis revealed that plant-produced DIII was processed properly and demonstrated specific binding to an anti-DIII monoclonal antibody that recognizes a conformational epitope. Furthermore, subcutaneous immunization of mice with 5 and 25 μg of purified DIII elicited a potent systemic response. This study provided the proof of principle for rapidly producing immunogenic vaccine candidates against WNV in plants with low cost and scalability.

Recombinant proteins are primarily produced from cultures of mammalian, insect, and bacteria cells. In recent years, the development of deconstructed virus-based vectors has allowed plants to become a viable platform for recombinant protein production, with advantages in versatility, speed, cost, scalability, and safety over the current production paradigms. In this paper, we review the recent progress in the methodology of agroinfiltration, a solution to overcome the challenge of transgene delivery into plant cells for large-scale manufacturing of recombinant proteins. General gene delivery methodologies in plants are first summarized, followed by extensive discussion on the application and scalability of each agroinfiltration method. New development of a spray-based agroinfiltration and its application on field-grown plants is highlighted. The discussion of agroinfiltration vectors focuses on their applications for producing complex and heteromultimeric proteins and is updated with the development of bridge vectors. Progress on agroinfiltration in Nicotiana and non-Nicotiana plant hosts is subsequently showcased in context of their applications for producing high-value human biologics and low-cost and high-volume industrial enzymes. These new advancements in agroinfiltration greatly enhance the robustness and scalability of transgene delivery in plants, facilitating the adoption of plant transient expression systems for manufacturing recombinant proteins with a broad range of applications.

Background: Public parks can be an important setting for physical activity promotion, but to increase park use and the activity levels of park users, the crucial attributes related to active park use need to be defined. Not only user characteristics and structural park attributes, but also characteristics of the surrounding neighborhood are important to examine. Furthermore, internationally comparable studies are needed, to find out if similar intervention strategies might be effective worldwide. The main aim of this study was to examine whether the overall number of park visitors and their activity levels depend on study site, neighborhood walkability and neighborhood income.

Methods: Data were collected in 20 parks in Ghent, Belgium and San Diego, USA. Two trained observers systematically coded park characteristics using the Environmental Assessment of Public Recreation Spaces (EAPRS) tool, and park user characteristics using the System for Observing Play and recreation in Communities (SOPARC) tool. Multilevel multiple regression models were conducted in MLwiN 2.25.

Results: In San Diego parks, activity levels of park visitors and number of vigorously active visitors were higher than in Ghent, while the number of visitors walking and the overall number of park visitors were lower. Neighborhood walkability was positively associated with the overall number of visitors, the number of visitors walking, number of sedentary visitors and mean activity levels of visitors. Neighborhood income was positively associated with the overall number of visitors, but negatively with the number of visitors being vigorously active.

Conclusions: Neighborhood characteristics are important to explain park use. Neighborhood walkability-related attributes should be taken into account when promoting the use of existing parks or creating new parks. Because no strong differences were found between parks in high- and low-income neighborhoods, it seems that promoting park use might be a promising strategy to increase physical activity in low-income populations, known to be at higher risk for overweight and obesity.

Background: Osteosarcoma is one of the most common bone cancers in children. Most patients with metastatic osteosarcoma die of pulmonary disease and limited curative therapeutic options exist for such patients. We have previously shown that PD-1 limits the efficacy of CTL to mediate immune control of metastatic osteosarcoma in the K7M2 mouse model of pulmonary metastatic disease and that blockade of PD-1/PD-L1 interactions can partially improve survival outcomes by enhancing the function of osteosarcoma-specific CTL. However, PD-1/PD-L1 blockade-treated mice eventually succumb to disease due to selection of PD-L1 mAb-resistant tumor cells. We investigated the mechanism of tumor cell resistance after blockade, and additional combinational therapies to combat resistance.

Methods: We used an implantable model of metastatic osteosarcoma, and evaluated survival using a Log-rank test. Cellular analysis of the tumor was done post-mortem with flow cytometry staining, and evaluated using a T-test to compare treatment groups.

Results: We show here that T cells infiltrating PD-L1 antibody-resistant tumors upregulate additional inhibitory receptors, notably CTLA-4, which impair their ability to mediate tumor rejection. Based on these results we have tested combination immunotherapy with α-CTLA-4 and α-PD-L1 antibody blockade in the K7M2 mouse model of metastatic osteosarcoma and show that this results in complete control of tumors in a majority of mice as well as immunity to further tumor inoculation.

Conclusions: Thus, combinational immunotherapy approaches to block additional inhibitory pathways in patients with metastatic osteosarcoma may provide new strategies to enhance tumor clearance and resistance to disease.

Contemporary vaccine development relies less on empirical methods of vaccine construction, and now employs a powerful array of precise engineering strategies to construct immunogenic live vaccines. In this review, we will survey various engineering techniques used to create attenuated vaccines, with an emphasis on recent advances and insights. We will further explore the adaptation of attenuated strains to create multivalent vaccine platforms for immunization against multiple unrelated pathogens. These carrier vaccines are engineered to deliver sufficient levels of protective antigens to appropriate lymphoid inductive sites to elicit both carrier-specific and foreign antigen-specific immunity. Although many of these technologies were originally developed for use in Salmonella vaccines, application of the essential logic of these approaches will be extended to development of other enteric vaccines where possible. A central theme driving our discussion will stress that the ultimate success of an engineered vaccine rests on achieving the proper balance between attenuation and immunogenicity. Achieving this balance will avoid over-activation of inflammatory responses, which results in unacceptable reactogenicity, but will retain sufficient metabolic fitness to enable the live vaccine to reach deep tissue inductive sites and trigger protective immunity. The breadth of examples presented herein will clearly demonstrate that genetic engineering offers the potential for rapidly propelling vaccine development forward into novel applications and therapies which will significantly expand the role of vaccines in public health.

Background: The current influenza vaccines are effective against seasonal influenza, but cannot be manufactured in a timely manner for a sudden pandemic or to be cost-effective to immunize huge flocks of birds. We propose a novel influenza vaccine composing a bacterial carrier and a plasmid cargo. In the immunized subjects, the bacterial carrier invades and releases its cargo into host cells where the plasmid expresses viral RNAs and proteins for reconstitution of attenuated influenza virus. Here we aimed to construct a mouse Poll-driven plasmid for efficient production of influenza virus. Results: A plasmid was constructed to express all influenza viral RNAs and proteins. This all-in-one plasmid resulted in 10(5)-10(6) 50 % tissue culture infective dose (TCID50)/mL of influenza A virus in baby hamster kidney (BHK-21) cells on the third day post-transfection, and also reconstituted influenza virus in Madin-Darby canine kidney (MDCK) and Chinese hamster ovary (CHO) cells. A 6-unit plasmid was constructed by deleting the HA and NA cassettes from the all-in-one plasmid. Cotransfection of BHK-21 cells with the 6-unit plasmid and the two other plasmids encoding the HA or NA genes resulted in influenza virus titers similar to those produced by the 1-plasmid method. Conclusions: An all-in-one plasmid and a 3-plasmid murine Poll-driven reverse genetics systems were developed, and efficiently reconstituted influenza virus in BHK-21 cells. The all-in-one plasmid may serve as a tool to determine the factors inhibiting virus generation from a large size plasmid. In addition, we recommend a simple and robust "1 + 2" approach to generate influenza vaccine seed virus.