DEVELOPMENT AND DELIVERY OF IMPROVED DNA VECTORS FOR GENE THERAPY AND DNA VACCINES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1011678
• Project No.: LAB94324
• Project Start Date: Dec 21, 2016
• Project End Date: Oct 31, 2020

Project Director
Cooper, RI.

Recipient Organization
LOUISIANA STATE UNIVERSITY
202 HIMES HALL
BATON ROUGE,LA 70803-0100

Performing Department
School of Animal Science

Non Technical Summary
The outcome of this project is two-fold. First, by developing DNA delivery methods that are capable of targeting specific tissues and cells, more efficient DNA uptake can be obtained which means more expression in the intended cells. By delivering only to target cells, costs are reduced since a lower volume of product is required to achieve the desired therapeutic levels and regulatory approval should be easier since there is little or no off-target incorporation of DNA. One of the regulatory concerns since DNA therapies were first attempted is the ability to demonstrate what the expression levels are and what dose is being delivered. While our approach does not remove all of these barriers, it significantly helps by limiting the cells expressing the DNA.Second, the DNA vaccines developed during this proposed research have significant bearing on production livestock agriculture and human medicine, if successful. By stably expressing multi epitope vaccines, it becomes possible to produce protection against a wide array of bacterial and viral pathogens not previously feasible to produce due to difficulty in growing, potential for adverse reactions, or inability to produce a vaccine that produces an immune response that is indistinguishable from an infected person or animal, i.e. Brucella vaccines. Because we are building a platform technology that has a "plug and play" feature, it becomes feasible to respond very rapidly to new diseases such as influenza, Ebola or Zika. The technology also allows rapid tweaking of DNA vaccines for different strains of bacterial pathogens and eliminates the possibility of creating antibiotic resistant bacteria, or "superbugs".Finally, because the end product does not require cold storage, we can produce vaccines for use in developing countries to improve human and livestock health. By being able to quickly and inexpensively respond to emerging diseases, it may become possible to one day contain diseases like Ebola or Zika before they pose a worldwide threat.

Animal Health Component

100%

Research Effort Categories

Basic

(N/A)

Applied

50%

Developmental

50%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3310	1090	75%
315	3510	1040	25%

Knowledge Area
311 - Animal Diseases; 315 - Animal Welfare/Well-Being and Protection;

Subject Of Investigation
3310 - Beef cattle, live animal; 3510 - Swine, live animal;

Field Of Science
1090 - Immunology; 1040 - Molecular biology;

Keywords

multi-epitope vaccines

dna vaccines

cell targeted dna delivery

Goals / Objectives
The purpose of this research is to develop an E. coli host strain that, upon induction, will remove the plasmid elements needed for replication and antibiotic resistance in the bacterial host. A DNA vaccine against Brucella abortus will be developed for proof-of-concept, but the technology has broad application to any DNA vaccine and gene therapy. Two goals will be addressed: 1) an E. coli host strain and vector system capable of removing plasmid replication elements in vitro to create pHalo-based vectors, and 2) development of transfection reagents capable of targeting cell receptors for more efficient, cell-specific transfection. Completion of the goals provides proof-of-concept for targeting a specific cell receptor with a ligand-transfection reagent to deliver pHalo devoid of E. coli replication elements. A review of the literature reveals no technology on the market similar to what is being developed for either goal. Combined, these technologies provide a safer, more efficient DNA transfer technology. A DNA vaccine against the human and livestock pathogen B. abortus is being designed for the purpose of proving the concept and potential international impact; due to the amount of research conducted in Dr. Elzer's laboratory, the tools are available to evaluate antigenic proteins produced in cell culture and study vaccine efficacy in an animal model. The proposed Anaplasma marginale vaccine will have direct impact on cattle operations in Louisiana. By providing an efficacious vaccine to eliminate the need for antibiotic treatment, farmers will have a tool to produce healthier cattle and eliminate the possibility of antibiotic resistant bacteria.An E. coli host strain and vector system will be completed that is capable of normal plasmid replication until late log phase. In late log phase, an inducible promoter will express a recombinase system that removes the antibiotic selection marker and the origin of replication, leaving only the gene-of-interest (GOI) in a "mini-halo" plasmid. The Food and Drug Administration (FDA) is developing policies on DNA vaccines and gene therapy products with a significant push towards "zero tolerance" on the inclusion of antibiotic selection markers and replicative elements. The concern is the horizontal gene transfer (HGT) of antibiotic resistance to other bacteria; and current methods to remove these elements are too costly, which can drive costs beyond what is affordable in the veterinary industry and developing countries. Second, two DNA transfection reagents will be developed containing receptor-specific ligands to increase plasmid delivery to a target cell. The expected outcome is twofold: 1) DNA uptake by non-target cells will be minimized; and 2) transfection efficiency will be increased. These transfection reagents will function in a similar manner to viral delivery vectors, targeting specific cell receptors with similar transfection efficiencies as viral vectors, yet retaining the non-immune stimulating feature of lipid-based transfection reagents.The specific objectives are:Objective 1.Complete the recombinase gene construct and incorporate into the E. coli chromosome by homologous recombinationClone the recombinase recognition sequences into a plasmid vector pHaloEstablish a growth curve for optimal timing of induction and recombinase expressionAnalyze pHalo production efficiencyGoal 1 will result in an E. coli based system capable of removing plasmid replication elements in situ and allow normal plasmid purification procedures.Objective 2.Attach peptide ligands to transfecting reagents with extensive transfection use in the literatureCompare peptide-bearing transfection reagents to those without peptide ligands to determine transfection efficiency with a reporter gene Goal 2 will provide a mechanism to deliver plasmid DNA or other nucleic acids to specific cells bearing the target receptor.Objective 3.Using a combination of computer modeling software, published research on Brucella and Anaplasma antigenic proteins, and collaborator knowledge, multi-epitope DNA vaccine(s) will be developed.Expression of the DNA vaccines will be tested in cell culture

Project Methods
MethodsMolecular cloning techniques will be used to construct DNA vaccines that have been designed and synthesized using DNA analysis software. Each vaccine will be tested for proper expression in cell culture as measured by ELISA and/or Western blot analysis. Ligand-transfection reagents will be tested for the ability to target specific cells using fluorescent imaging, FACS, and DNA uptake using real time PCR. If additional funding is obtained, animal trials will be conducted and an immune response measure using ELISA and Western blot analysis.

Progress 12/21/16 to 10/31/20

Outputs
Target Audience:The target audience includes livestock producers and dairy farmers in Louisiana, the U.S., and worldwide. The vaccines resulting from this project will be of particular importance to small animal producers and farmers that could quickly lose their profitability if an animal dies or requires an expensive, long-term treatmentor reproductive capabilities of valuable seedstock are inhibited. The human and veterinary pharmaceutical industries would also have significant interest in DNA vaccines developed during this project. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Three graduate students are being trained on this project: two Ph.D. students and one M.S. To date, 24 undergraduates have received course credit for undergraduate research as they have been trained using various components of this project including cell culture, molecular biology, and animal care in an experimental environment. How have the results been disseminated to communities of interest?Class discussions,committee meetings, seminars, and local poster sessions. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1.1 & 1.2 After a series of complications, the recombinase system is nearly complete; the final product is being evaluated at the time of this report. The pHalo vector has been completed, and vaccine candidates have been cloned into it. Objective 2 Since the initiation of this project, commercially available transfection reagents have become available. Specifically, reagents are available that allow targeting of dendritic cells by having a mannose ligand attached to the reagent. Subcutaneous injectioninto quail and goatsis currently underway as part of the Ph.D. dissertations. Objective 3.1 Most of our efforthas been focused on this objective. Using bioinformatics, twoBrucella abortus,threeSalmonella javiana, and twoAnaplasma marginalevaccines were designed and constructed. Each of these vaccines were tested in cell culture and a goat and/or quail model to determine which provided epitope expression recognizedby commerically available convalescent sera. Based on data obtained from these experiments, a vaccine candidate from each pathogen was selected; and the vaccine components cloned into the pHalo vector. Once the recombinase strain is completed, pHalo + vaccine will be produced as described in Objective 1. To date, each selected vaccine has been demonstrated to induce an immune response in model species. Challenge studies will take place in 2021/2022.

Publications

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:The target audience includes livestock producers and dairy farmers in Louisiana, the U.S., and world-wide. The vaccines resulting from this project will be of particular importance to small producers and farmers that could quickly lose their profitability if an animal dies or requires an expensive, long term treatment, or reproductive capabilities of valuable seedstock are inhibited. The human and veterinary pharmaceutical industries would also have significant interest in DNA vaccines developed during this project. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Three graduate students are being trained on this project: two Ph.D. students and one M.S. To date, 18 undergraduates have received course credit for undergraduate research as they have been trained using various components of this project including cell culture, molecular biology, and animal care in an experimental environment. How have the results been disseminated to communities of interest?Class discussions and committee meetings What do you plan to do during the next reporting period to accomplish the goals?Work with Objectives 1 & 2 will continue in 2020. Completion of these objectives has been slow because they are either part of graduate student projects or being used to teach research techniques to undergraduates; there is no research associate available for this project. Objective 3 will continue with a larger scale goat trial for B. abortus and quail trial for S. javiana.

Impacts
What was accomplished under these goals? Objective 1 is still in progress, with approximately 2/3 of the vector system complete. Objective 2 is in progress with initial attempts of ligands being linked to the transfecting reagent. Preliminary cell culture data suggests we were successful in transfecting cells, and we will being focusing additional efforts in this area in the coming year. Objective 3 is complete. DNA vaccines for S. javiana, B. abortus, and A. marginalewere constructed and tested in cell culutre. Based on theseresults, the vaccines for B. abortus and S. javiana were tested in a pilot animal trial using goats; two vaccines were tested for B. abortus, with one proving superior. The superior vaccine will be tested in a larger animal trial beginning in the spring of 2020. Data for S. javiana is still being evaluated.

Publications

• Type: Other Status: Other Year Published: 2019 Citation: Smith, Caitlyn, A. Edwards, and R. Cooper. 2019. Detection of Immune Response for S. javiana DNA Vaccine. LSU Discover Undergraduate Research poster

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:The target audienceincludes livestock producers and dairy farmers in Louisiana, the U.S., and in foreign countries. The vaccines resulting from this project will be of particular importance tosmall producers and farmers that could quickly lose their profitability if an animal dies or requires an expensive, long term treatment, or reproductive capabilities of valuable seedstock are inhibited. The human and veterinary pharmaceutical industries would also have significant interest in DNA vaccines developed during this project. Changes/Problems:To further investigate our vaccine candidates, we have added in vivo studies using goats and quail. What opportunities for training and professional development has the project provided?Three graduate students are being trained on this project; two Ph.D. students and one M.S. To date, 18 undergraduates havereceived course credit for undergraduate research as they have been trained using various components of this project including cell culture, molecular biology, and animal care in an experimental environment. How have the results been disseminated to communities of interest?One undergraduate research poster was presented at LSU Discovery Dayfor Undergraduate Research. This presentation focused on DNA vaccine construction and the impact potential of DNA vaccines in human and veterinary medicine. What do you plan to do during the next reporting period to accomplish the goals?Objective 1. It is anticipated that the E. coli host strain needed for DNA vaccine replication will be completed during the next reporting period. Once complete, experiments will be conducted to determine if the system works as designed to remove the antibiotic resistance gene and plasmid replication components. Objective 2. The peptide ligand::transfection reagent will be tested in cell culture and compared to transfection reagents without a peptide ligand in order to compare efficiency of DNA delivery into a targeted cell. We will also test a new transfection reagent we are developing; if it functions correctly, it will more efficiently target cells of the mammalian immune system in vivo. Objective 3. Cell culture experiments with the B. abortus vaccines will be repeated to confirm the first results and the cell culture results from A. marginale vaccines will be tested and analyzed.

Impacts
What was accomplished under these goals? Progress was made on each objective during the reporting period. Specific details for each objective are described below. Objective 1. Progress was made but is slow due to the complexity of the system being built and because some components are being used to teach undergraduates the tools of molecular biology. Most of the components have been constructed and are in the process of being assembled. Completion and testing is expected during the 2018/2019 reporting period. Objective 2.1. Completed. Peptide ligands were successfully attached to a transfection reagent. This reagent has not been tested yet due to retirement of the employee assigned to this project. Objective 3.1. This objective has been completed;the vaccines have been constructed andsequence verified. Objective 3.2. This objective is in progress and has been partially completed. The vaccines for B. abortus have been tested in cell culture, an ELISA optimized, and results obtained from one of the vaccines indicatesit is expressing epitopes recognized by B. abortus convalescent serum from bovines. For A. marginale, an ELISA has been developed and optimized; but the cell culture experiments are still in progress at this point.

Publications

• Type: Other Status: Other Year Published: 2018 Citation: Construction and testing of three DNA vaccines against�S. javiana. A. Edwards, B. Faucheaux, and R. Cooper. LSU Discovery Day Undergraduate Research Poster Presentation.

Progress 12/21/16 to 09/30/17

Outputs
Target Audience:The target audienceincludes vaccine companies,veterinarains, undergraduate students, graduate students, and governement regulatory agencies with an interest in novel vaccine and gene therapy approaches. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Both undergraduate and graduate students (M.S. and Ph.D.) are participating in this project to learn DNA vaccine design and how they stimulate cell-mediated immunity; how to use bioinformatics programs; to master techniques in molecular biology; and how to culture, transfect, and select cell culture clones. Components of this work are also used in undergraduate genetics lectures to teach students about vaccine research being conducted in the LSU Agricultural Center. How have the results been disseminated to communities of interest?Two undergraduate posters were presented in an undergraduate research symposium called LSU Discovery Day. What do you plan to do during the next reporting period to accomplish the goals?Final synthesis of the components needed to create the host strain will be assembled and transfected into the E. coli host strain in order to create a bacterial host capable of replicating aDNA vaccine vector and upon induction of the promoter, express enzymes to remove the antibiotic resistance marker and recircularize the vaccine vector. Once completed, each vaccine vector produced will be tested for antigen expression in cell culture; both media and cell lysates will be analyzed by either Western blot or ELISA using convalescent serum to the respective pathogen for the vaccine.

Impacts
What was accomplished under these goals? We are developing a DNA vaccine platform that addresses all three areas described in the project summary. During this reporting period, several multi-epitope vaccine candidates were synthesized and cloned into sequencing vectors. We are in the process of cloning each vaccine into the final expression vector pHalo. In addition, the first cell-specific ligand::transfection reagent was synthesized and will be tested in early 2018. Last, each of the components needed to create the E. coli host strain to remove the antibiotic resistance marker has been synthesized and cloned. The final assembly is in progress, and the host strain should be complete in early 2018 and ready for testing the vaccine candidates.

Publications

• Type: Theses/Dissertations Status: Published Year Published: 2017 Citation: McGee, M. 2017 DESIGN AND HUMORAL ANALYSIS OF TWO EPITOPE-BASED BRUCELLA ABORTUS DNA VACCINES. Master's Thesis submitted to Louisiana State University