New design may produce more effective Salmonella-based vaccine

August 7, 2013

The bacterial pathogen Salmonella has a notorious capacity for infection. Last year alone, according to the Center for Disease Control, various species of Salmonella caused multistate disease outbreaks linked with contaminated peanut butter, mangoes, ground beef, cantaloupe, poultry, tuna fish, small turtles and dry dog food.

The troublesome invader, however, can be turned to human advantage. Through genetic manipulation, the species S. Typhi can be rendered harmless and used in vaccines in order to prevent, rather than cause illness. Salmonella vaccine infographic Download Full Image

In new research, reported in the Journal of Bacteriology, lead author Katie Brenneman and her colleagues describe efforts to improve the effectiveness of a Recombinant Attenuated Salmonella Vaccine (RASV) by modifying its ability to survive the hostile environment of the stomach.

“Even though wild-type strains of Salmonella are quite capable of surviving the acidic environment of the stomach, it is surprisingly difficult to deliver a live Salmonella vaccine orally,” Brenneman says. “Many vaccines have mutations that leave them especially vulnerable to low pH, which means a large proportion of the vaccine cells are killed before they reach the intestine, and thus are unable to do their job of delivering vaccine targets to the immune system. We’re trying to compensate for that increased acid sensitivity by increasing expression of the normal acid resistance systems.” 

The group demonstrated experimental strategies to restore acid resistance in several Salmonella vaccine strains, thereby improving their ability to survive low pH conditions in the stomach. The improved survival rate allows more of the bacterial cells to continue their infection sequence, colonizing intestinal tissues and generating a strong immune response.

Further, the acid resistant vaccine strains may behave more like unmodified Salmonella, which are cued by low pH conditions to prepare for the later stages of the infection process by up-regulating a key suite of genes involved in host interactions. These factors, the authors suggest, may significantly improve the effectiveness of Salmonella vaccines.

Co-authors of the study include ASU Biodesign Institute researchers Crystal Willingham, Wei Kong, Roy Curtiss III and Kenneth L. Roland.

At the Biodesign Institute’s Center for Infectious Diseases and Vaccinology at ASU, researchers have been harnessing Salmonella’s impressive ability to infiltrate human tissues and stimulate immune responses, producing Salmonella-based vaccines targeting a range of illnesses.

The center is under the direction of Roy Curtiss III, whose team has been genetically modifying the pathogen in efforts to produce a new breed of safe, efficient and cost- effective vaccine.

Salmonella vaccines offer great potential in meeting growing needs for effective protection against existing and emergent threats. One such vaccine – designed by the Curtiss group and currently in Phase I FDA trials – targets infant pneumonia, a disease that continues to kill some 2 million people per year, many of them in the developing world. Other RASV’s are in various stages of development.

Such vaccines are attractive for a number of reasons. They can typically be produced much more cheaply than conventional vaccines, they may be delivered orally rather than through injection and can confer long-term immunity without the requirement of a subsequent booster dose. Further, Salmonella powerfully stimulates both cellular and humoral immunity, producing a robust, systemic response in the vaccine recipient.

The basic idea behind RASVs is to genetically retool the Salmonella bacterium in such a way that it retains its strong, immunogenic properties without causing illness. It is then outfitted with antigens for the particular disease the vaccine is designed to protect against. This Trojan-horse method introduces the disease antigens hidden in the Salmonella carrier, which then stimulate the immune responses. 

But as the authors of the current study explain, the promising technique – potentially applicable for vaccines against virtually any pathogen – is not without its challenges. One of the most significant hurdles concerns the ability of Salmonella to survive the harsh environment of the stomach, where highly acidic (low pH) conditions prevail.

Naturally occurring, unmodified Salmonella have evolved sophisticated strategies of acid tolerance and acid resistance that allow them to survive the stomach environment. By contrast, modified Salmonella strains cultured in the laboratory are weakened or attenuated to improve their safety. The process has the negative effect of greatly reducing Salmonella’s acid tolerance.

A number of features allow normal Salmonella to survive low pH conditions. Two of the most important have been studied in some detail. The first is known as the acid tolerance response (ATR) and the second, the arginine decarboxylase acid resistance system. The latter of these mechanisms is not expressed in Salmonella cultured for vaccine use and the remaining ATR system is often insufficient to protect bacterial cells from stomach acid.

One approach to the problem has been to protect the vaccine strain from low pH conditions by shrouding it in an enteric capsule. Alternately, vaccines have been administered in conjunction with an antacid to lower stomach pH, (typically with sodium bicarbonate).

These strategies improve the survival of vaccine strains but have the disadvantage of depriving Salmonella of encountering the low pH environment, which acts to signal the bacteria that they have entered the host environment. These signals stimulate the upregulation of genes to help Salmonella survive the next phase of infection in the intestine, where it is threatened by short-chained fatty acids, antimicrobial peptides and osmotic stress. Further, induction of normal acid tolerance response improves Salmonella’s ability to invade epithelial cells in the intestine.

In the current study, researchers attempted to restore acid tolerance in modified Salmonella at pH levels of 3 and 2.5 in order to overcome the loss of tolerance imposed by three common gene mutations used for RASVs. To accomplish this, a hybrid version of the arginine decarboxylase acid resistance system was created. This system was not only capable of inducing acid resistance in cultured Salmonella cells, but could be tightly controlled by means of a special promoter, triggered by the presence of the sugar rhamnose.

Use of the rhamnose promoter to induce acid resistance allows cultured cells to be prepared to withstand the low pH rigors of the stomach. Following the initial stages of infection, the bacterial cells expend their storehouse of rhamnose and their acid resistance is then rapidly downregulated.

Experiments confirmed that the rhamnose-regulated system could indeed rescue Salmonella from exposure to low pH conditions and that it provided bacterial cells with an equivalent degree of protection from acidic environments to the native arginine decarboxylase system.

The three acid unadapted mutants used in the study all showed significant improvement in survival at pH conditions of 3 and 2.5. The results open the door to the efficient and cost-effective creation of highly acid resistant vaccine strains that can exhibit fine-grained control under a rhamnose promoter and can be produced on demand. Allowing vaccine strain exposure to low gastric pH should further improve performance, by triggering virulence genes necessary for subsequent survival and colonization in the intestine.

“In future studies, we will need to validate that this system, or other similar systems under construction, will improve the immunogenicity of an RASV,” says co-author Ken Roland. “This work is ongoing, but I can tell you that we have preliminary data supporting the idea that our rhamnose-regulated arginine decarboxylase system can significantly enhance the immunogenicity and protective immunity of an RASV.”

Written by:

Richard Harth,
Biodesign Institute science writer

Media contact:

Joe Caspermeyer

Manager (natural sciences), Media Relations & Strategic Communications


New grant advances ASU microscopy imaging initiative

August 7, 2013

Peering through a homemade instrument – toy-like by today’s standards – the Dutch tradesman Antony van Leeuwenhoek (1632-1723) first observed a dizzying menagerie of lifeforms, invisible to the naked eye. Since then, scientists have steadily refined the field of microscopy, achieving spectacular results at ever-tinier scales.

At Arizona State University’s Biodesign Institute, Nongjian (NJ) Tao has been designing advanced microscopy methods with the ambitious aim of capturing molecular-scale phenomena in living systems. The new techniques, which combine multiple imaging modalities, are poised to revolutionize the study of biology and the development of new drugs. Professor N.J. Tao Download Full Image

“To study the dynamics of individual molecules in living systems, extremely high resolution alone is not enough,” Tao says. “You also need the ability to image and record very fast processes as they occur.”

Tao is the recipient of a new $1.6 million grant from the Gordon and Betty Moore Foundation for research titled, "Label-free imaging and tracking of single protein-protein interactions." The three-year project calls for the design of a novel microscopy system based in part on a phenomenon known as plasmonic resonance.

The new microscope will allow researchers to not only observe a specimen’s form at a remarkably minute scale, but also investigate chemical reactions and charge-related properties of living systems. The microscopy techniques employed will permit these molecular spectacles to be imaged with greatly enhanced contrast and unprecedented temporal resolution.

Tao directs Biodesign’s Center for Bioelectronics and Biosensors. In previous research, the group exploited plasmonic imaging to examine single cells, cell organelles, viruses and nanoparticles. The new grant calls for an extension of this work in order to image the dynamics of single protein interactions in living systems – a feat never before accomplished.

Proteins are essential components in all life processes and play a central role in the maintenance of health and the onset of disease. A more thorough understanding of their multifarious activities is crucial. Until now however, studying the subtle dynamics of proteins in their native state – including their binding properties – has been beyond the ability of imaging technology.

Over the course of the three-year project, the research team will develop a fast and low-noise plasmonic imaging system capable of resolving fine structure, as well as following the transport of sub-cellular features with startling precision, with temporal resolution in the sub-millisecond range.

Getting closer

A variety of sophisticated techniques have been applied in order to extend the range and versatility of modern microscopy, from traditional optical methods to scanning probe techniques, opening up previously inaccessible realms. Such methods have succeeded at imaging structures down to the single molecule level, i.e., fractions of a nanometer – an enormous achievement.

These techniques have been invaluable for the advance of biological research and medical diagnostics, however some critical tasks have remained out of reach. One of these is the direct imaging of single molecules within living systems – for example, a live cell.

Optical microscopy methods, including fluoresce and confocal imaging, have produced stunning images at high resolution. Nevertheless, the resolving power of such instruments has been constrained by the diffraction limit of light, which dictates that spatial resolution is limited to approximately half the wavelength of the incident light used to illuminate the sample.

From a practical standpoint this means that conventional optical microscopy cannot resolve features smaller than 200-300 nanometers – too small to visualize subcellular structures, much less, proteins, which are typically less than 10 nanometers.

Electron microscopy is one method used to overcome the diffraction limit of light, but the technique requires involved sample preparation and is not suitable for imaging living systems like cells. Electrons cannot penetrate the native aqueous environment of most biological matter and energetic electrons can damage biological samples.

On the other hand, scanning probe techniques, like atomic force microscopy, which use a probe to scan a biological sample line by line, and which are applicable in aqueous environments, are not fast enough to capture dynamic processes at the molecular level, which occur at very high speed.

A new look

Tao’s technique relies on a phenomenon known as surface plasmon resonance. When polarized light strikes a specially prepared surface coated with a thin metallic film, free electrons (or plasma) absorb incident photons and convert them into a surface plasmon wave that propagates like a water wave near the surface of the metal film.

When a sample such as a living cell, virus or nanoparticle interacts with the plasmon wave, it disrupts it, thereby causing a measurable change in light reflectivity. These alterations in reflectivity can be converted into an image.

Plasmonic imaging in general is limited to samples near the surface of the metal film with an image depth of around 200 nm. While this limitation prevents imaging of whole cell bodies, it is easily capable of imaging proteins, viruses, dendrites of neurons and cell organelles.

Using a newly invented technique known as P-EIM (plasmonic electrical impedence imaging), image depth can be significantly increased. P-EIM also improves imaging contrast and importantly, allows charge-related properties of biological samples to be imaged.

The P-EIM imaging method relies on the fact that the surface plasmonic signal is sensitively dependent on the surface charge density, which can be measured optically. P-EIM permits very rapid, non-invasive impedance imaging in living systems and unlike normal surface plasmon resonance, can monitor a thick biological sample such as an entire cell body.

“Recent developments in optical microscopy have created exciting new ways to investigate the structure and function of living cells, but directly ‘seeing’ molecules interacting in live cells remains a major challenge,” said Dr. Gary Greenburg, program officer with the Gordon and Betty Moore Foundation. “We believe the development of the  Plasmonic-based Electrochemical Impedance Microscope will deliver novel, label-free imaging capabilities that do not currently exist and will have a major impact on our fundamental understanding of how cells work.”

A related technique, known as P-ECM (plasmonic-based electrochemical current imaging), measures electrochemical current density from an optical signal produced by surface plasmon resonance, thereby allowing precision imaging of electrochemical reactions. The rapid and non-invasive nature of P-ECM permits the imaging of catalytic reactions in single nanoparticles for the first time.

The new microscope system therefore enables the simultaneous imaging of gross morphology, chemical reactions and charge-related properties (including charged molecules or ions) using a single imaging system. The technique provides high spatial resolution, as well as unprecedented temporal resolution capable of imaging very fast molecular phenomena, as surface plasmons can respond to light on femtosecond time scales.

Part of the new grant project will involve tracking the movement of mitochondria in a living cell. Tao stresses that the new microscope’s unrivalled spatial and temporal resolution will help provide new insights into the interactions of motor proteins between mitochondria and microtubules, among many other molecular-scale events.

Using new, ultrafast cameras capable of recording over a million frames per second, plasmonic imaging with microsecond time resolution will be possible. Further, the technique is compatible with conventional bright field and fluorescence imaging.

Thus, a single instrument can combine the imaging strengths of conventional and plasmon-based techniques, opening the door to intense investigations of life processes occurring in their native state at the most intimate scale.

Written by:

Richard Harth,
Biodesign Institute science writer

Media contact:
Joe Caspermeyer

Manager (natural sciences), Media Relations & Strategic Communications