Simpler technique yields antibodies to a range of infectious agents

ASU study promises to deliver new insights into the structure and function of membrane proteins of critical importance for medicine

February 25, 2016

Researchers hope to develop vaccines, therapeutics and new diagnostic tests for a broad range of diseases. To accomplish this, they will need to gain a much better understanding of a critical class of biological components. Known as surface membrane proteins, these vital ingredients in the disease process form a structurally and functionally diverse assemblage of enormous complexity.

In a new study, Debra Hansen, a research professor at Arizona State University’s Biodesign Institute, explores an innovative means of investigating membrane proteins produced by a pair of highly pathogenic organisms. The research team showed that DNA-based genetic immunization, using a device known as a gene gun, could successfully express membrane proteins in mice and induce the animals to produce a range of critical antibodies to bacterial and viral targets. Antibodies, (seen in green, red and orange) bind with specific membrane proteins present on the cell surface. Graphic by Jason Drees for the Biodesign Institute. Antibodies, (seen in green, red and orange) bind with specific membrane proteins present on the cell surface. Graphic by Jason Drees/Biodesign Institute Download Full Image

“We learned that our new process of antibody production is incredibly efficient. If the membrane protein is naturally immunogenic, we easily generated high levels of antibodies using the genes alone,” Hansen (pictured below) said. “Rather than laboriously purifying the membrane protein and attempting to maintain the proper protein structure within detergents prior to immunization, we let the immunized host do the work for us.”

The new study also describes a method for expressing and purifying membrane proteins in a test tube and examining their binding activities with specific antibodies in blood extracted from gene-immunized mice.

Conventionally, producing an immune response to a foreign protein requires purification of the protein, which is then injected into an animal. The process is cumbersome, challenging and time consuming. In the current study, an immune response is instead produced by directly introducing a gene encoding the protein of interest.

Two of the membrane proteins produced in the study were also successfully introduced into membranes of the bacterium E. coli, through the process of recombinant DNA. Use of the specific antibodies present in blood from gene-immunized mice demonstrated for the first time that both membrane proteins could be recombinantly expressed in a live organism, correctly fold into proper 3-D structures and migrate to the membranes within E. coli. These results now facilitate the structural determination of these two critical virulence proteins.

portrait of ASU professor Debra HansenThe research, which appears in the current issue of the Nature Publishing Group journal Scientific Reports, promises to deliver new insights into the structure and function of membrane proteins of critical importance for medicine.

On the surface

Membrane proteins are implicated in innumerable functions in living organisms including cell signaling and communication, energy conversion and utilization, molecular transport and catalysis. Due to their involvement in a range of diseases, they have recently become primary targets for a new range of therapeutics. Indeed, more than 50 percent of all therapeutic targets are membrane proteins. The number is expected to rise as more is learned about membrane protein structure and function.

Despite their important role as the molecular interface in host/pathogen interactions as well as drug/cell relationships, membrane proteins account for less than 1 percent of the 100,000 unique protein structures presently catalogued. This is largely due to the serious challenges involved in producing, purifying and determining the structures of membrane proteins. Hansen and her colleagues outline new strategies to produce antibodies — specialized proteins produced naturally by the immune system in response to pathogens or other threatening biological agents.

In the current study, they describe the use of a gene gun to introduce DNA information into a mouse. The handheld device uses a burst of gas to propel gold particles impregnated with circular DNAs known as plasmids into the skin in mice. The gold particles are known as micronanoplexes. The gene gun technology was pioneered and developed by Stephen A. Johnston, co-director of the Biodesign Center for Innovations in Medicine.

The genetic material introduced via the gene gun is taken up by mouse dendritic cells and translated into membrane protein in dermal tissues and lymph nodes. The mouse immune system responds by producing specific antibodies capable of binding to the membrane proteins. While the basic technique of genetic immunization has been in use for some time, the study marks the first description of the broad applicability of this approach to membrane proteins, as well as the first application of DNA-gold micronanoplexes to stimulate antibody production.

Bacterial and viral menaces examined

Results showed that genetic immunization successfully produced antibodies specific to 12 out of 17 membrane proteins from two Biosafety Level 3 pathogens: Francisella tularensis and African swine fever virus (ASFV). F. tularensis causes the disease tularemia. It is a widely studied infectious pathogen notorious for its ability to invade numerous cell types and cleverly evade the immune system. It is one of the most pathogenic bacteria on Earth, capable of causing a fatal infection with as few as 10 cells. African swine fever virus is carried by arthropods. Infection in pigs causes a lethal and untreatable hemorrhagic disease that has devastated swine populations in areas of Africa and Eastern Europe.

Investigations of endogenous disease proteins produced by these organisms are difficult, requiring specialized safety facilities and protocols due to the potential danger they pose to researchers. The new method described permits the production of these pathogens’ membrane proteins and associated antibodies through DNA-based approaches, permitting the safe handling of biological material without risk of infection.

Once antibodies to specific membrane proteins have been produced in the mouse, the group sought to characterize the resulting mouse blood or sera. To do this, a new system known as in vitro translation in the presence of hydrophobic magnetic beads (IVT-HMB) was developed. Here, a small quality of membrane protein is produced in a test tube, simultaneously extracted using hydrophobic beads, then screened against the sera extracted from gene-immunized mice. Detection of a resulting signal in two types of diagnostic tests or assays (ELISA and Western blot) established the presence in the mouse sera of antibodies specific to each membrane protein. The IVT-HMB method represents a powerful streamlining of the production of membrane proteins, precluding the arduous process of isolation and purification traditionally required.

Stepping-stone to protein structures

Using X-rays to image tiny crystals of proteins is a powerful method to determine detailed protein structure, but the technique faces many challenges, including the difficulty of producing and purifying proteins that assemble properly. The current research marks a starting point for further structural characterization of membrane proteins, using such techniques as cryo-EM and X-ray crystallography.

Petra Fromme, a co-author of the new study and director of the Biodesign Institute’s Center for Applied Structural Discovery, highlights the power of the new research: “The range of antibodies produced through techniques like genetic immunization opens the door to high-resolution molecular images of important membrane proteins,” she said. “The resulting antibodies assist structural determination in a variety of important ways, identifying properly assembled proteins, helping to induce proteins to assemble with other proteins into well-ordered crystals and stabilizing or trapping proteins in active states that can be imaged using X-rays.”

In the next phase of research, the group plans to produce monoclonal antibodies using the same immunization process. These are essential as co-crystallization binding factors or ligands, used for the structural determination of membrane proteins via X-ray crystallography. The authors further note that the monoclonal antibodies produced through genetic immunization techniques offer attractive candidates for future therapeutics against a broad range of diseases.

This study was a combined effort of faculty in the Biodesign Institute, including Debra Hansen from the Center for Innovations in Medicine (CIM), Center for Applied Structural Discovery (CASD) and the School of Molecular Sciences (SMS), Kathryn Sykes from CIM and Petra Fromme from CASD and SMS, along with their teams of researchers and students, including: research scientists Mark Robida, Andrey Loskutov and Tien Olson, researchers Felicia Craciunescu, John-Charles Rodenberry and Hetal Patel and graduate student Xiao Wang from CIM and postdoc Katerina Dörner from SMS.

All of the necessary clones (plasmid DNAs) for applying this approach are available through the DNASU Plasmid Repository, which is housed in Biodesign’s Center for Personalized Diagnostics.

This work was funded by the NIH (NIGMS) under the PSI:Biology program, as part of the MPID (Membrane Proteins in Infectious Diseases) U54 grant, directed by Petra Fromme.

Richard Harth

Science writer, Biodesign Institute at ASU


Innovative ASU-created device traces chemicals affecting human, environmental health

February 25, 2016

Every hour, a multitude of chemicals complete their use life in homes, agricultural fields and industries and flood into the environment. The ultimate fate of these compounds is often poorly understood, as are the risks they may pose to humans and the ecosystems that are essential for our survival as a species.

In a new study, a multi-disciplinary, multi-institutional team of researchers headed by Rolf Halden, director of the Center for Environmental Security at Arizona State University’s Biodesign Institute, tracks the course of a family of widely used pesticides known as fiproles. These halogenated chemicals have been identified as an emerging contaminant, recently linked to the worldwide die-off of pollinating insects, particularly honeybees. An in situ water contaminate sampling device in use, looks like a metal tube in a river. An earlier version of the in situ sampling device. The lower portion of the tubular instrument is submerged in sediment while the upper portion is exposed to pore water. The IS2B tool can register contaminants at very low detection limits for a broad spectrum of contaminants, ranging from fully water-soluble to highly sorptive and hydrophobic Download Full Image

To properly assess the levels of fiprole contamination in the environment, Halden’s team invented a new device, constructed at the Biodesign Institute. Known as the IS2B, the tool is a kind of mobile laboratory or pod for performing precision analysis on sampled water and sediment. The technology offers improved accuracy of measurement compared with existing methods as well as greater versatility and cost-effectiveness.

“Health risks from pollution are dependent not necessarily on the absolute quantity of toxins present but rather on what fraction of the total pollutant mass is accessible for uptake by living organisms. The patent-pending IS2B device is designed to tell apart and quantify these two important quantities,” said Halden.

Halden is joined by professor Nancy Denslow and collaborators from the Department of Physiological Sciences and Center for Environmental and Human Toxicology, University of Florida, Gainesville. The research appears in the advanced online issue of the Nature Publishing Group journal Scientific Reports.

Hazards unseen

Human and aquatic lifeforms face increasing threats from chemical contamination. The United States Environmental Protection Agency (USEPA) estimates that 10 percent of the sediments located in domestic lakes, rivers and harbors are contaminated with potentially harmful chemicals. It is therefore essential to carry out environmental sampling to properly evaluate the degree of hazard and design remediation strategies, where needed.

Soils, sediments and water resources can bind chemicals to varying degrees, making them available to microbes, plants, wildlife and humans. Yet, few of the chemicals in daily use have been properly assessed for safety. Researchers like Halden and Denslow hope to measure the capacity of potentially hazardous chemicals to be absorbed by living organisms, a quantity known as their bioavailability.

Certain chemicals are known to evade degradation, persisting in the environment for decades. Among these tenacious pollutants are hydrophobic compounds, which are highly durable and resistant to breakdown. Such chemicals can be tricky to measure and track, due to their ability to permanently sequester in pore spaces between sedimentary particles. Often, they are not degraded during the normal process of wastewater treatment.

Measurement of bulk water and sedimentary pore water can provide something like an environmental blood test, informing researchers about the presence and relative risk of chemicals of concern in samples. Accurate environmental sampling is often challenging, however. Many contaminants occur in low concentrations in the environment, yet may seriously affect living organisms due to uptake and long-term storage, a process termed bioaccumulation. Current methods of sample collection, preparation, and analysis may under- or overestimate the actual chemical mass available to cause harm in exposed animals and humans.

An in situ water contaminate sampling device in use, the image above is the previous version, and the image below is an older version

Newer version of the device, known as the
IS2B. The tool is a kind of mobile laboratory
or pod for performing precision analysis on
sampled water and sediment.

Sifting for chemicals

The in situ sampling/bioavailability tool (IS2B) is a specialized instrument providing simultaneous sampling of contaminant levels in bulk and sedimentary pore water at previously unattainable detection limits.

The IS2B contains integrated multi-channel pumps, which simultaneously draw surface water and sediment pore water into the active sampling device. Then, the water is pushed at high and low flow rates through an array of filters and adsorption media. When in use, the lower half of the tubular instrument is buried in sediment, while the upper portion is exposed to pore water.

The water samples can be stored in the device or readily expelled into the bulk water. Due to unlimited access to pore and bulk water during use, the IS2B tool can register contaminants at very low detection limits for a broad spectrum of contaminants, ranging from fully water-soluble to highly sorptive and hydrophobic. 

The device allows for direct determination of bioavailability and ultimately seeks to replace or minimize the use and exposure of animals for toxicity assessment. Samples may be collected over arbitrary time periods and the method can evaluate both dissolved and particulate contaminants in both free-flowing surface water and more stagnant pore water in the sediment.

A suspect emerges

Fipronil, the focus of the new study, is a broad-spectrum halogenated pesticide that has recently been banned in the European Union for most agricultural uses. Fipronil’s use in the United States, however, remains widespread. The chemical is the active ingredient in many pesticide formulations used in both urban and agricultural settings, for such applications as termite treatment, seed treatments, and to protect turf from pest infestation.

Scientists have been alarmed and perplexed by the rapid collapse of honeybee populations. Serious efforts to identify the culprit have produced conflicting data. Honeybees are vital participants in the complex web of life, pollinating roughly 80 percent of all flowering plants. One third of all agricultural production depends directly on bee pollination. Mite infestations, viral pathogens and the impact of pesticides all have been implicated in honeybee colony collapse disorder, as the epidemic is known.

The new study sampled water and sediment extracted from three locations in a wetland in the Southwestern United States. The wetland examined receives outfall from a waste treatment plant, adjacent to agricultural fields. The results of IS2B in situ monitoring of fiprole concentrations were compared with those obtained by more cumbersome and time-demanding conventional benchtop laboratory analysis.

Results showed close agreement between IS2B data and the current gold standard of measurement techniques. Further, the study was able to identify the chemical fipronil-desulfinyl occurring at one location in trace amounts that evaded detection by conventional sampling, highlighting the benefits of continuous in situ analysis for capturing transient fluxes of chemicals at levels much lower than previously attainable. Use of the IS2B also reduces the chances of sample contamination from handling in the laboratory facility, as the majority of sample processing steps are conducted in place at the sampling site.

Another important benefit of the device is its ability to collect samples continuously for extended periods of time, from days to several weeks. This makes possible the detection of short-term fluctuations in chemical loading, such as illegal dumping of process streams into surface waters. As such, information gathered with the IS2B is vital to environmental compliance reporting, modeling and risk assessment for biota and humans.