I am a genetic engineer who has had the extraordinary opportunity to practice my art in the laboratory for over 20 years. My years in the lab have been some of the most exhilarating times of my professional career, and have led to significant advances in both vaccine development and innovative methods for testing the immunogenicity of candidate vaccine strains. These efforts have resulted in multiple patents awarded both in the United States, as well as internationally. In the last few years, I have stepped away from bench work, focusing more intently on mentoring, teaching, speaking at professional meetings, and the challenges of securing funding for further development of live vaccines. Here, I will summarize how I became interested in molecular biology, the accomplishments for which I am most pleased, the impact of these discoveries on the field of vaccine development, and my efforts to share my enthusiasm in translational research with students, doctoral candidates, and post-doctoral investigators.
My interest in molecular biology and genetic engineering goes back to my first introduction to DNA in a high school biology class. When I first learned that all of life can be encoded using just four nucleotides, I was hooked. This astonishing elegance in nature has fascinated me to this day, and with the advent of molecular tools that could be used to actually change what microorganisms encode and when they express these genes, I have managed to amuse myself for several decades now. However, I learned very early on that I am a creature of translational applications. I have never been overly enamored with facts per se unless I could figure out a way to exploit that information to solve a problem. Without the “how” and “why”, I just cannot seem to be able to focus on the “what” and the “when.” Indeed, when I attempted my first course in genetics in college, I ended up dropping out of the class twice because I just could not get overly excited about crossover frequencies and probabilities. Not willing to walk away from such abject failure, I took the class one last time, and as the old cliché goes “the rest is history.” This particular genetics class was taught from a mechanistic point of view, stressing the molecular aspects of genetic events, how they occur, and what drives these processes. This was the world that I decided to spend my life trying to pursue.
Through the years, I have had the somewhat unique opportunity of working in both industry and in academia, both as a technician and later as a Ph.D. I first worked as a technician, fresh out of college, at a small company called Bethesda Research Laboratories, purifying restriction enzymes and assisting in the teaching of classes in DNA sequencing, which was an emerging technique in the early eighties. It was through one of these classes taught at the University of Maryland Baltimore that I met Dr. James Kaper, who offered me the opportunity to carry out basic research in his laboratory as a Research Assistant. During this period in Dr. Kaper's lab, I was perfectly content to carry out research into the molecular characterization of cholera toxin expressed by the enteric pathogen Vibrio cholerae. I had absolutely zero intention to return to school because I did not see the need to do so; I was participating in research, without the stress of classes, exams, and writing, and I could see myself working in this arena for many years to come. However, with encouragement from Dr. Kaper, I eventually enrolled in graduate school and was awarded my Ph.D. in 1991. Again, anxious to use the new knowledge that I had gained and not just continue to study, I chose not to pursue post-doctoral training, instead accepting a position as a Research Scientist at MedImmune, Inc. At MedImmune, I managed to stay at the lab bench, carrying out research into the development of plasmid-based expression systems for use in Bacille Calmette-Guérin (BCG) vaccines. During this time, I became very interested in the use of attenuated vaccine strains as live carrier strains, for presentation of foreign antigens to the human immune system as multivalent vaccines. I was approached in 1993 by Dr. Myron M. Levine at the Center for Vaccine Development (CVD), University of Maryland Baltimore, who offered me a faculty appointment at the CVD to carrying out research in the nascent field of developing live carrier vaccines derived from attenuated Salmonella enterica serovar Typhi typhoid vaccine strains.
It was during the interview process prior to returning to the University of Maryland Baltimore that I was given a valuable piece of advice which I did not appreciate at the time. When asked why I wanted to return to the University, I answered that I just wanted to do research in the laboratory; at the time, I had no interest in running my own laboratory or developing research projects. I just wanted to do the work. But the gentleman who was interviewing me advised that if I planned to be in research for any length of time, I would eventually want to ask my own questions and then pursue the answers myself. Years later, I finally realized that he was right, and this is when I started creating my own unique engineering technologies to solve problems that interested me.
Upon accepting Dr. Levine's offer to come to the Center for Vaccine Development, I focused the genetic engineering skills that I had acquired at MedImmune to develop plasmid-based expression systems for use in attenuated S. Typhi live carriers. At the time I started this work, the genetic technology available for this type of work had mainly been developed for use in attenuated Salmonella Typhimurium strains. Attempts to adapt these genetic tools for use in S. Typhi produced unstable vaccines, and refinement of such strains was further complicated by the lack of an acceptable animal model for testing the immunogenicity of candidate S. Typhi-based vaccines. Therefore, our team set out to establish a new murine model of intranasal immunization which proved to be extremely efficient, leading to three important publications by Barry et al (1996), Galen et al. (1997), and Pickett et al. (2000) that firmly established this approach as the model of choice for pre-clinical testing of attenuated S. Typhi-based vaccine strains.
After our team established the murine intranasal model of immunogenicity, I was able to turn my attention to the vexing problem of maintaining plasmids expressing foreign antigens in attenuated S. Typhi vaccines. I found multicopy expression plasmids to be inherently unstable in S. Typhi, being quickly lost in the absence of drug selection, which would ultimately lead to the failure of such vaccines when tested both in the murine model and eventually in humans. The first iteration of a potential solution to the plasmid stability problem took advantage of naturally occurring plasmid maintenance systems found on very low copy plasmids encoding resistance to antibiotics. These systems encoded a toxin which was toxic to S. Typhi, but also encoded an antitoxin to block toxicity; the beauty of the system was that the antitoxin was inherently unstable and required constant synthesis from the plasmid to avoid lethality. Plasmid loss would immediately remove the resulting bacterium from the population, thus ensuring a population of vaccine organisms all carrying the expression plasmid and presumably expressing the desired foreign protein(s) in vivo. Unfortunately, it became apparent that spontaneous mutations arising in the gene encoding the toxin could quickly lead to plasmid loss, and indeed this mechanism is suspected to have been observed in a recent clinical trial of an attenuated Shigella live vector in which the toxin-antitoxin system was employed (Kotloff et al. unpublished).
The second iteration of a plasmid maintenance system solved this problem and ultimately removed the need for use of antibiotics to select for plasmids after introduction into attenuated S. Typhi vaccines. This system relies on successful deletion from the chromosome of a gene encoding an essential protein, and insertion of this gene instead onto a low copy expression plasmid to again remove the possibility of plasmid loss. I chose to target the ssb gene, encoding the single-stranded binding protein (SSB) which is absolutely essential for DNA replication, recombination, and repair. Implementation of this approach proved highly successful, leading to development of a candidate S. Typhi-based live vector vaccine against anthrax which was published in 2010. Patents have also been issued for this technology both in the United States and worldwide.
During the period in which plasmid maintenance systems were being developed, I also began to realize that should we be successful in genetically stabilizing expression plasmids, we would eventually have to address the possibility that stable expression of potentially toxic but highly immunogenic vaccine antigens within attenuated S. Typhi vaccines might ultimately lead again to vaccine failure. I chose to solve this problem by developing a novel antigen secretion system in which foreign proteins fused to the carboxyl-terminus of this carrier protein can be exported out to the surface of the live vector, or even expelled into the extracellular medium. This approach was used in the development of vaccines against malaria caused by Plasmodium falciparum, as well as further refinements to our live vector-based anthrax vaccine, and reports of this work were published in peer-reviewed journals in 2008 and 2009 respectively. Multiple national and international patents have now been awarded for this export technology from 2007 through 2010.
I have now attempted to merge all of this expression technology together in the development of multivalent live carrier-based vaccines against infections caused by Clostridium difficile and Acinetobacter baumannii. I have continued to improve upon the strategy of presenting foreign antigens on the surface of live carrier vaccines to improve immunogenicity and protective efficacy. I realized that if the foreign vaccine antigens to be targeted were outer membrane proteins, these proteins could naturally be expressed on the surface of live carrier strains without any further engineered surface expression strategies. However, as the field of molecular biology is always evolving and refining itself, new technologies and observations always present themselves to those who vigilantly scour the literature. And, sure enough, it was recently reported that overexpression of a protein called PagL induces the formation of outer membrane vesicles in Salmonella. The reason behind this involves removal of acyl groups from lipid A, which also reduces the reactogenicity of these vesicles. This simple observation now opens up the possibility of using attenuated S. Typhi as a “vaccine platform”, expressing and exporting immunogenic and protective outer membrane proteins from unrelated pathogens to the surface of the vaccine, such that expression of PagL can be used to deliver these antigens as purified outer membrane vesicle vaccines. These novel recombinant purified vesicles can in turn be used in combination with the live carriers in a heterologous prime-boost immunization strategy to dramatically improve vaccine efficacy. Patents on this technology are currently pending.
In addition to my research efforts, I have also been involved for at least the last decade in mentoring doctoral candidates and post-docs, as well as teaching various classes in plasmid and molecular biology. I find teaching to be a very rewarding experience in which I can reach students and inspire them to realize that molecular biology offers them a whole new world of exploration in which remarkably simple techniques can be turned into vaccines that could one day improve public health. As I experienced myself in college, students seem to appreciate it when investigators relate basic molecular mechanisms to explain the reasons behind emerging medical challenges such as the alarming rise in bacteria acquiring resistance to most known antibiotics. With Ph.D. candidates, I value the back-and-forth intellectual sparring that occasionally occurs with doctoral candidates not willing to accept current dogma. I truly enjoy the enthusiastic responses of students as they begin to understand what is actually involved in the conduct of research that applies the power of genetic engineering; it is easy to miss the fact that, if successful, engineers have the absolute privilege of applying their craft to the creation of novel organisms that previously did not exist in nature until engineered for the first time. I would do my job for free if I could afford to do so. Getting out of bed in the morning to see what is in the incubator will be a difficult habit to replace in my retirement. Needless to say, I will be dragged kicking and screaming into that unpleasant but hopefully distant possibility.
About James Galen. Dr. Galen is Professor of Medicine and Chief of the Salmonella Vaccine Section at the University of Maryland School of Medicine. He received his PhD (1991) from the same institution, and then left academics to work at MedImmune (1991–3), where he worked on expression systems for BCG vaccines. Upon returning to the University of Maryland Baltimore, his work has focused for the last several decades on the development of enteric vaccines and bacterial vectors. Dr. Galen has made seminal contributions to the development of Salmonella-based carriers for delivery of heterologous antigens and plasmid-based systems for antigen expression. He has co-authored >40 articles and book chapters, and has secured multiple national and worldwide patents for his work.