Network


Latest external collaboration on country level. Dive into details by clicking on the dots.

Hotspot


Dive into the research topics where James A. Williams is active.

Publication


Featured researches published by James A. Williams.


Biotechnology Advances | 2009

Plasmid DNA Vaccine vector design: impact on efficacy, safety and upstream production

James A. Williams; Aaron E. Carnes; Clague P. Hodgson

Critical molecular and cellular biological factors impacting design of licensable DNA vaccine vectors that combine high yield and integrity during bacterial production with increased expression in mammalian cells are reviewed. Food and Drug Administration (FDA), World Health Organization (WHO) and European Medical Agencies (EMEA) regulatory guidances are discussed, as they relate to vector design and plasmid fermentation. While all new vectors will require extensive preclinical testing to validate safety and performance prior to clinical use, regulatory testing burden for follow-on products can be reduced by combining carefully designed synthetic genes with existing validated vector backbones. A flowchart for creation of new synthetic genes, combining rationale design with bioinformatics, is presented. The biology of plasmid replication is reviewed, and process engineering strategies that reduce metabolic burden discussed. Utilizing recently developed low metabolic burden seed stock and fermentation strategies, optimized vectors can now be manufactured in high yields exceeding 2 g/L, with specific plasmid yields of 5% total dry cell weight.


Vaccine | 2009

Improved antibiotic-free DNA vaccine vectors utilizing a novel RNA based plasmid selection system

Jeremy Luke; Aaron E. Carnes; Clague P. Hodgson; James A. Williams

To ensure safety, regulatory agencies recommend elimination of antibiotic resistance markers from therapeutic and vaccine plasmid DNA vectors. Here, we describe the development and application of a novel antibiotic-free selection system. Vectors incorporate and express a 150 bp RNA-OUT antisense RNA. RNA-OUT represses expression of a chromosomally integrated constitutively expressed counter-selectable marker (sacB), allowing plasmid selection on sucrose. Sucrose selectable DNA vaccine vectors combine antibiotic-free selection with highly productive fermentation manufacturing (>1g/L plasmid DNA yields), while improving in vivo expression of encoded proteins and increasing immune responses to target antigens. These vectors are safer, more potent, alternatives for DNA therapy or vaccination.


Biotechnology and Applied Biochemistry | 2006

Inducible Escherichia coli fermentation for increased plasmid DNA production.

Aaron E. Carnes; Clague P. Hodgson; James A. Williams

Bacterial plasmids are the vectors of choice for DNA vaccines and short‐term gene therapeutics. Growing plasmid DNA by microbial (Escherichia coli) fermentation is usually combined with alkaline lysis/chromatography methods of purification. To date, typical plasmid fermentation media and processes result in yields of 100–250 mg of plasmid DNA/l of culture medium, using standard high‐copy pUC origin‐containing plasmids. In order to address this initial and yield‐limiting upstream step, we identified novel fermentation control parameters for fed‐batch fermentation. The resulting fermentation strategies significantly increased specific plasmid yield with respect to cell mass while enhancing plasmid integrity and maintaining supercoiled DNA content. Fed‐batch fermentation yield exceeding 1000 mg of plasmid DNA/l was obtained after reduction of plasmid‐mediated metabolic burden during growth, and yields up to 1500 mg of plasmid DNA/l have been achieved with optimized plasmid backbones. Interestingly, by inducing high plasmid levels after sufficient biomass accumulation at low temperature and restricted growth, cells were able to tolerate significantly higher plasmid quantities than cells grown by conventional processes. This 5–10‐fold increase in plasmid yield dramatically decreases plasmid manufacturing costs and improves the effectiveness of downstream purification by reducing the fraction of impurities.


Biotechnology and Bioengineering | 2009

Generic plasmid DNA production platform incorporating low metabolic burden seed-stock and fed-batch fermentation processes

James A. Williams; Jeremy Luke; Sarah Langtry; Sheryl Anderson; Clague P. Hodgson; Aaron E. Carnes

DNA vaccines have tremendous potential for rapid deployment in pandemic applications, wherein a new antigen is “plugged” into a validated vector, and rapidly produced in a validated, fermentation—purification process. For this application, it is essential that the vector and fermentation process function with a variety of different antigen genes. However, many antigen genes are unpredictably “toxic” or otherwise low yielding in standard fermentation processes. We report cell bank and fermentation process unit operation innovations that reduce plasmid‐mediated metabolic burden, enabling successful production of previously known toxic influenza hemagglutinin antigen genes. These processes, combined with vector backbone modifications, doubled fermentation productivity compared to existing high copy vectors, such as pVAX1 and gWiz, resulting in high plasmid yields (up to 2,220 mg/L, 5% of total dry cell weight) even with previously identified toxic or poor producing inserts. Biotechnol. Bioeng. 2009;103: 1129–1143.


Journal of Virology | 2011

Coexpressed RIG-I Agonist Enhances Humoral Immune Response to Influenza Virus DNA Vaccine

Jeremy Luke; Gregory G. Simon; Jonas Söderholm; John S. Errett; J. Thomas August; Michael Gale; Clague P. Hodgson; James A. Williams

ABSTRACT Increasing levels of plasmid vector-mediated activation of innate immune signaling pathways is an approach to improve DNA vaccine-induced adaptive immunity for infectious disease and cancer applications. Retinoic acid-inducible gene I (RIG-I) is a critical cytoplasmic double-stranded RNA (dsRNA) pattern receptor required for innate immune activation in response to viral infection. Activation of RIG-I leads to type I interferon (IFN) and inflammatory cytokine production through interferon promoter stimulator 1 (IPS-1)-mediated activation of interferon regulatory factor 3 (IRF3) and NF-κB signaling. DNA vaccines coexpressing antigen and an expressed RNA (eRNA) RIG-I agonist were made, and the effect of RIG-I activation on antigen-specific immune responses to the encoded antigen was determined. Plasmid vector backbones expressing various RIG-I ligands from RNA polymerase III promoters were screened in a cell culture assay for RIG-I agonist activity, and optimized, potent RIG-I ligands were developed. One of these, eRNA41H, combines (i) eRNA11a, an immunostimulatory dsRNA expressed by convergent transcription, with (ii) adenovirus VA RNAI. eRNA41H was integrated into the backbone of DNA vaccine vectors expressing H5N1 influenza virus hemagglutinin (HA). The resultant eRNA vectors potently induced type 1 IFN production in cell culture through RIG-I activation and combined high-level HA antigen expression with RNA-mediated type I IFN activation in a single plasmid vector. The eRNA vectors induced increased HA-specific serum antibody binding avidity after naked DNA intramuscular prime and boost delivery in mice. This demonstrates that DNA vaccine potency may be augmented by the incorporation of RIG-I-activating immunostimulatory RNA into the vector backbone.


Recent Patents on Biotechnology | 2007

Plasmid DNA Manufacturing Technology

Aaron E. Carnes; James A. Williams

Today, plasmid DNA is becoming increasingly important as the next generation of biotechnology products (gene medicines and DNA vaccines) make their way into clinical trials, and eventually into the pharmaceutical marketplace. This review summarizes recent patents and patent applications relating to plasmid manufacturing, in the context of a comprehensive description of the plasmid manufacturing intellectual property landscape. Strategies for plasmid manufacturers to develop or in-license key plasmid manufacturing technologies are described with the endpoint of efficiently producing kg quantities of plasmid DNA of a quality that meets anticipated European and FDA quality specifications for commercial plasmid products.


Cell | 1975

Adenovirus recombination: Physical mapping of crossover events

James A. Williams; Terri Grodzicker; Phillip A. Sharp

Abstract Recombinants have been isolated from crosses of temperature-sensitive mutants of two adenovirus serotypes whose DNAs differ in their cleavage patterns with restricting endonucleases. Recombinant and parental genomes were dissected with restriction enzymes and the resulting fragments compared. From the results one can align the adenovirus genetic and physical maps.


Cell | 2016

HIV-1 Neutralizing Antibodies with Limited Hypermutation from an Infant

Cassandra A. Simonich; Katherine L. Williams; Hans Verkerke; James A. Williams; Ruth Nduati; Kelly K. Lee; Julie Overbaugh

HIV-1 broadly neutralizing antibodies (bnAbs) develop in a subset of infected adults and exhibit high levels of somatic hypermutation (SHM) due to years of affinity maturation. There is no precedent for eliciting highly mutated antibodies by vaccination, nor is it practical to wait years for a desired response. Infants develop broad responses early, which may suggest a more direct path to generating bnAbs. Here, we isolated ten neutralizing antibodies (nAbs) contributing to plasma breadth of an infant at ∼1 year post-infection, including one with cross-clade breadth. The nAbs bind to envelope trimer from the transmitted virus, suggesting that this interaction may have initiated development of the infant nAbs. The infant cross-clade bnAb targets the N332 supersite on envelope but, unlike adult bnAbs targeting this site, lacks indels and has low SHM. The identification of this infant bnAb illustrates that HIV-1-specific neutralization breadth can develop without prolonged affinity maturation and extensive SHM.


Cell | 1975

The location of the genes coding for hexon and fiber proteins in adenovirus DNA

Vivien Mautner; James A. Williams; Phillip A. Sharp; Terri Grodzicker

A serological analysis has been made of the capsid antigens hexon and fiber from 17 Ad5-Ad2+ND1 recombinants that enables us to determine the phenotype of the recombinants. By correlation of this data with the genetic and physical maps of the adenovirus genome, obtained by recombination and restriction endonuclease analysis, the genes coding for the hexon and fiber have been assigned to specific locations on the adenovirus DNA.


Vaccine | 2013

Vector Design for Improved DNA Vaccine Efficacy, Safety and Production.

James A. Williams

DNA vaccination is a disruptive technology that offers the promise of a new rapidly deployed vaccination platform to treat human and animal disease with gene-based materials. Innovations such as electroporation, needle free jet delivery and lipid-based carriers increase transgene expression and immunogenicity through more effective gene delivery. This review summarizes complementary vector design innovations that, when combined with leading delivery platforms, further enhance DNA vaccine performance. These next generation vectors also address potential safety issues such as antibiotic selection, and increase plasmid manufacturing quality and yield in exemplary fermentation production processes. Application of optimized constructs in combination with improved delivery platforms tangibly improves the prospect of successful application of DNA vaccination as prophylactic vaccines for diverse human infectious disease targets or as therapeutic vaccines for cancer and allergy.

Collaboration


Dive into the James A. Williams's collaboration.

Top Co-Authors

Avatar

Kelly K. Lee

University of Washington

View shared research outputs
Top Co-Authors

Avatar

Cassandra A. Simonich

Fred Hutchinson Cancer Research Center

View shared research outputs
Top Co-Authors

Avatar

Julie Overbaugh

Fred Hutchinson Cancer Research Center

View shared research outputs
Top Co-Authors

Avatar

Hans Verkerke

University of Washington

View shared research outputs
Top Co-Authors

Avatar
Top Co-Authors

Avatar

Katherine L. Williams

Fred Hutchinson Cancer Research Center

View shared research outputs
Top Co-Authors

Avatar

Long Gui

University of Washington

View shared research outputs
Top Co-Authors

Avatar

Phillip A. Sharp

Massachusetts Institute of Technology

View shared research outputs
Top Co-Authors

Avatar

Shiu-Lok Hu

University of Washington

View shared research outputs
Top Co-Authors

Avatar

Terri Grodzicker

Cold Spring Harbor Laboratory

View shared research outputs
Researchain Logo
Decentralizing Knowledge