Many research findings from his laboratory have been published in highly ranked journals, including Nature, Cell and Science. Importantly, Walter Fiers was always thinking ahead for possible applications of the research findings from his lab. His visionary research lies at the basis of recombinant gene expression for the production of biologicals such as insulin, industrial enzymes and certain vaccines.  This is one of the most important technological accomplishments on which modern pharmaceutical industry and many research laboratories now thrive. Walter Fiers and his collaborators in the Laboratory of Molecular Biology at Ghent University pioneered this field.

Walter Fiers (° 31^st of January 1931, Ieper, Belgium) graduated as an Engineer of Chemistry and Agricultural Sciences at Ghent University in 1954. In 1960 Walter obtained the diploma of “Agrégé” for Higher Education in Biochemistry, notably 3 years before his PhD diploma that he obtained in 1963 under the supervision of Laurent Vandendriessche (Laboratory of Physiological Chemistry, Faculty of Medicine, Ghent University). By that time, Walter Fiers had already worked for 3 years as a scientist in two outstanding research laboratories in the USA. In the summer of 1960 he and his wife moved to California where he joined the lab of Robert L. Sinsheimer at the renowned California Institute of Technology (CalTech, Pasadena, California, USA) as a Research Fellow in Biology, supported by a fellowship from the Rockefeller Foundation and the NFWO (Nationaal Fonds voor Wetenschappelijk Onderzoek). In the early 1960s it was known that the genome of the “minute” Escherichia coli bacteriophage fX174 consisted of a single stranded DNA chain of approximately 5500 nucleotides. Although phage genetics was in vogue because it allowed to map genes on a DNA genome, still very little was known about the physico-chemical structure of DNA molecules. By using a series of exo- and endonucleases combined with analytical velocity sedimentation experiments, Walter demonstrated that the genome of phage fX174 is a circular molecule. This conclusion represented a breakthrough finding and the experimental evidence that Walter had gathered to prove this point was published in 3 research articles, all submitted on the same day, that appeared in the Journal of Molecular Biology in 1962 (1-3). In the fall of 1962, Walter Fiers moved to Madison to join the laboratory of H. Gobind Khorana at the Institute of Enzyme Research at the University of Wisconsin (USA). In the lab of G. Khorana, who (in 1968) received the Noble Price in Medicine and Physiology, together with Robert W. Holley and Marshall W. Nirenberg, for their contribution to the elucidation of the genetic code, Walter Fiers purified and characterized an exonuclease from Lactobacillus acidophilus. Dr. Khorana had developed a method to synthesise oligodexoynucleotides, which was far from evident at that time, and it was decided at some point to feed the exonuclease Walter was characterizing different types of these polynucleotides. It turned out that the enzyme was picky and could not hydrolyse polyadenylic and polyuridylic acid, whereas thymidine oligonucleotides were readily turned into single nucleotides by the enzyme. The work was published under the heading “Studies of Polynucleotides” in the Journal of Biological Chemistry (4, 5). In one of these papers the authors stated: “The possibility of using the phosphodiesterase for the sequence determination in polynucleotides is discussed” (4). This is exactly what Walter Fiers had in mind after his return to Belgium in 1963: use nucleases to determine the sequence of a polynucleotide. His aim, however, was extremely ambitious: to determine the sequence of the complete genome of a bacteriophage. According to many of his peers this was an impossible project and it was difficult to raise the necessary funding to start the project. Walter Fiers proved them wrong.

From 1963 to 1967 Walter was appointed assistant professor at the Faculty of Agricultural Sciences and in 1967 he became an associate professor at the Faculty of Sciences of Ghent University where he established the Laboratory of Molecular Biology. His lab was next to that of Marc Van Montagu and Jozef Shell in the Ledeganckstraat in Ghent, which created an inspiring and enthusiastic scientific atmosphere. Walter recruited very talented PhD students and his team set out to determine the genome sequence of the Escherichia coli bacteriophage MS2. This phage was chosen because it provided a means to isolate high amounts of intact RNA as it is sheltered by the phage coat, and because the MS2 genome serves as a mRNA for this virus. The experimental approach was to chop the radiolabelled RNA genome of MS2 into pieces with RNase T1 or pancreatic RNAse and then isolate the resulting oligonucleotides. Two-dimensional gel electrophoresis, paper chromatography and treatment with exonucleases were subsequently used to deduce the primary nucleotide sequence of these oligonucleotides. Needless to say that the project required a long term vision and perseverance. A typical PhD thesis in those days in the Fiers’ lab described the sequence of 5 to 10 pentanucleotides of phage MS2. Over time, overlapping sequences were spotted, which slowly, piece by piece, allowed to reconstitute the genome sequence of MS2. In 1972 Min Jou et al., published the complete nucleotide sequence of the MS2 coat protein gene (6). This was the first time that the complete sequence of any gene coding for a protein was determined. Moreover, the work allowed to confidently annotate codon usage because the peptide sequence of the MS2 coat protein had just been determined by Joel Vandekerckhove and Marc Van Montagu (7). In 1975 Walter Fiers’ team published the sequence of the MS2 maturation protein (also known as the A-protein) (8). This paper was followed in 1976 by the publication, again in Nature, of the “Complete nucleotide sequence of bacteriophage MS2 RNA: primary and secondary structure of the replicase gene” (9). This was truly a landmark achievement: it was the first ever published complete genome. Much more, however, was still to come.

In the beginning of the 1970s, Walter Fiers had established mammalian cell culture in his laboratory. So it was possible to culture a mammalian virus and, of course, to sequence the genome of such a virus. The team in Ghent focussed on Simian Virus 40 (SV40), which is a non-enveloped DNA virus with a circular genome of 5224 base pairs. The interest in SV40 was inspired by the fact that SV40 had the potential to cause tumours in inoculated animals. Unravelling the genome sequence could thus shed light on the molecular basis of tumour formation. The approach was to purify DNA restriction fragments and sequence these using the chemical degradation method developed by Maxam and Gilbert. After a series of publications that appeared in Cell and Nature between 1976 and 1978 on parts of the SV40 genome sequence, the complete nucleotide sequence of this eukaryotic DNA virus was published by Fiers et al., in 1978 (10-12). In addition, the SV40 sequencing work in Ghent contributed to the discovery of RNA splicing in eukaryotes (13). Still today, many plasmids used for expression of proteins in eukaryotic cells contain genetic elements of the SV40 genome (e.g. ori, promoters, polyadenylation signals).

At the end of the 1970s the lab of Walter Fiers was not only world famous for its contributions to nucleotide sequencing technologies but also for the cloning of genes. The lab was the first to clone and sequence a cDNA copy of human interferon beta, a cytokine with multiple activities and best known for its antiviral effect. This endeavour was the result of a concerted effort with Eric De Clercq from the KULeuven and Jean Content from the Institut Pasteur in Brussels (14). Being an engineer by training, Walter Fiers quickly realized the practical and medical implications of trying to produce interferon beta as well as other cytokines with potential medical applications in heterologous expression systems. This would, in principle, make it possible to produce large quantities of these proteins and, if desired, to make mutants with altered properties. Erik Remaut, who had joined the Fiers laboratory in 1966 had been sent to the USA by Walter and, on his return in 1977, introduced a prokaryotic expression system based on the powerful, leftward promoter of phage lambda in the lab. This allowed the team to prove that Escherichia coli could indeed produce biologically active human interferon beta in a practical, inducible way (15).

Many mammalian signalling molecules and secreted proteins, including most cytokines, are post-translationally modified. Glycosylation, for example, is often extremely important for the correct folding and function of eukaryotic membrane and secreted proteins. Despite the many benefits of bacterial recombinant protein expression systems, they are severely limited in their capacity to accomplish such modifications. Walter Fiers’ lab helped to solve this limitation by establishing robust heterologous protein expression systems in yeast and fungi, both capable of glycosylation (16, 17). This pioneering work showed that it was possible to use eukaryotic microbial systems, that had already proven their value in industrial processes for decades, now also for the production of medically applicable biologicals that closely matched their natural counterparts.

However, the zest of the Laboratory for nucleotide sequencing remained high and in the late 1970s Walter Fiers set out to determine the primary structure of the major antigenic spike protein of influenza A viruses by sequencing the corresponding viral coding regions. In humans, influenza viruses cause the flu, an important respiratory disease that all too often causes health problems in the very young and the elderly. In addition, pigs, horses as well as many bird species are hosts of influenza A viruses. It was known that influenza A viruses can swiftly escape from vaccine-induced or naturally acquired immunity by a process known as antigenic drift. This means that the antigenicity of the viral hemagglutinin and neuraminidase, the 2 main spike proteins of influenza viruses, changes over time. As a result, people can get the flu many times in life. However, how this antigenic drift was controlled genetically was unclear at the time. Moreover, influenza A viruses were known to occasionally cause pandemics, a fearful event during which an influenza virus with an antigenically highly different hemagglutinin emerges and spreads (notorious examples are the 1918 “Spanish flu” and the “Hong Kong flu” of 1968). In 1980 two publications appeared from the Fiers lab that not only reported the complete sequence of the hemagglutinin gene of the H3N2 influenza virus that had caused the Hong Kong flu pandemic of 1968, but also elucidated the mechanism of antigenic drift (18, 19). The comparison of the hemagglutinin sequence of an H3N2 virus that circulated in 1975 with that of its 1968 ancestor, revealed that this viral antigen had acquired a selected number of mutations that were confined to antigenic sites on the surface of the protein. These mutations left the biological function of the hemagglutinin intact but altered the sequence of highly immunogenic loops of the protein (19). One year later, the Molecular Biology lab in Ghent also explained how pandemic influenza viruses can emerge. The sequence analysis of the hemagglutinin of an avian influenza virus that had been isolated in 1963, unambiguously showed that this viral gene had been acquired by a human virus, which allowed it to cause a pandemic in 1968 (20).

In the 1980s, DNA sequencing became a mainstream method that was implemented worldwide in many molecular biology laboratories. As a result, an ever increasing number of sequences of genes from all kinds of organisms, including influenza viruses, became public. This information confirmed the remarkable sequence diversity of the hemagglutinin (and neuraminidase) of human influenza viruses but also revealed that one part of the virus that is accessible on the surface, is remarkably conserved. This conserved part is the extracellular domain of the matrix 2 membrane protein, in short M2e. Could this conserved domain, that was seemingly difficult for the virus to change, be targeted by a recombinantly produced vaccine? If so, surely, one would expect very broad (“universal”) protection against influenza A viruses by such a vaccine. Walter Fiers proposed to his collaborators to genetically graft M2e on a sturdy virus-like particle that could be massively produced in an economical bacterial expression system, developed in his own lab. The experimental vaccine was successfully produced, protected vaccinated mice against influenza A virus infection and protection could be transferred by immune serum, as reported in 1999 in Nature Medicine (21). In collaboration with a pharmaceutical company, this M2e-based vaccine was later successfully tested in a phase I clinical trial: the vaccine was safe and induced M2e-specific antibodies in the volunteers. Still today, many research laboratories and biotech companies are investigating ways to implement M2e as an important component for the as yet to come universal influenza vaccine. Indeed, we are not there yet, because it may require a combination of conserved influenza antigens, and thus a complicated vaccine, to be able to induce a potent, durable cross-protective immune response against human influenza. Walter Fiers’ most recent scientific achievement was the idea to develop an antiviral molecule that would target M2e. The anti-influenza agent that Walter conceived is a bi-specific single chain antibody-derived molecule that selectively recruits T cells to the influenza A virus-infected cells. As a result, these cells are swiftly eliminated, which prevents virus spread and saves the host. The idea worked in vitro and in an experimental mouse model for influenza, findings that were published in 2017 (22).

The outstanding contributions of Walter Fiers to the field of microbiology are also reflected in some of the many prestigious scientific prices that were awarded to him: the Francqui Award (Francqui Foundation, Belgium – 1976), the Dr. Beijerinck Gold Medal for Virology (Royal Dutch Academy of Sciences, The Netherlands – 1986), the Artois-Baillet Latour Prize (Belgium – 1989), the Carlos J. Finlay Prize (UNESCO Prize for Microbiology, including Immunology, Molecular Biology and Genetics – 1989) and the Robert Koch Prize (Robert Koch-Stiftung, Bonn, Germany – 1991).

Walter Fiers officially retired as a professor of Molecular Biology at the faculty of Sciences at the University of Ghent in 1996. However, he remained active as a free-lance strategic adviser in his former laboratory until 2016, during which time he focused his attention on the research in the Molecular Virology group. His impact on modern biotechnology and microbiology can hardly be overestimated.

Walter Fiers passed away on July 28th, 2019.

Xavier Saelens, Wim Declercq & Erik Remaut

Sources

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2. Fiers W, Sinsheimer RL. 1962. The structure of the DNA of bacteriophage phi-X174. II. Thermal inactivation. Journal of molecular biology 5:420-423.
3. Fiers W, Sinsheimer RL. 1962. The structure of the DNA of bacteriophage phi-X174. III. Ultracentrifugal evidence for a ring structure. Journal of molecular biology 5:424-434.
4. Fiers W, Khorana HG. 1963. Studies on Polynucleotides. 23. Enzymic Degradation. An Exonuclease from Lactobacillus Acidophilus R26. B. Stepwise Degradation of Oligonucleotides. The Journal of biological chemistry 238:2789-2796.
5. Fiers W, Khorana HG. 1963. Studies on Polynucleotides. Xxii. Enzymic Degradation. An Exonuclease from Lactobacillus Acidophilus R26. A. Purification, Properties, and Substrate Specificity. The Journal of biological chemistry 238:2780-2788.
6. Min Jou W, Haegeman G, Ysebaert M, Fiers W. 1972. Nucleotide sequence of the gene coding for the bacteriophage MS2 coat protein. Nature 237:82-88.
7. Vandekerckhove J, Francq H, Van Montagu M. 1969. The aminoacid sequen of the coat protein of the bacteriophage MS-2 and localization of the amber mutation in the coat mutants growing on a su+3 suppressor. Archives internationales de physiologie et de biochimie 77:175-176.
8. Fiers W, Contreras R, Duerinck F, Haegmean G, Merregaert J, Jou WM, Raeymakers A, Volckaert G, Ysebaert M, Van de Kerckhove J, Nolf F, Van Montagu M. 1975. A-protein gene of bacteriophage MS2. Nature 256:273-278.
9. Fiers W, Contreras R, Duerinck F, Haegeman G, Iserentant D, Merregaert J, Min Jou W, Molemans F, Raeymaekers A, Van den Berghe A, Volckaert G, Ysebaert M. 1976. Complete nucleotide sequence of bacteriophage MS2 RNA: primary and secondary structure of the replicase gene. Nature 260:500-507.
10. Contreras R, Rogiers R, Van de Voorde A, Fiers W. 1977. Overlapping of the VP2-VP3 gene and the VP1 gene in the SV40 genome. Cell 12:529-538.
11. Fiers W, Contreras R, Haegemann G, Rogiers R, Van de Voorde A, Van Heuverswyn H, Van Herreweghe J, Volckaert G, Ysebaert M. 1978. Complete nucleotide sequence of SV40 DNA. Nature 273:113-120.
12. Van de Voorde A, Contreras R, Rogiers R, Fiers W. 1976. The initiation region of the SV40 VP1 gene. Cell 9:117-120.
13. Haegeman G, Fiers W. 1978. Evidence for ‘splicing’ of SV40 16S mRNA. Nature 273:70-73.
14. Derynck R, Content J, DeClercq E, Volckaert G, Tavernier J, Devos R, Fiers W. 1980. Isolation and structure of a human fibroblast interferon gene. Nature 285:542-547.
15. Derynck R, Remaut E, Saman E, Stanssens P, De Clercq E, Content J, Fiers W. 1980. Expression of human fibroblast interferon gene in Escherichia coli. Nature 287:193-197.
16. Contreras R, Carrez D, Kinghorn JR, van den Hondel CA, Fiers W. 1991. Efficient KEX2-like processing of a glucoamylase-interleukin-6 fusion protein by Aspergillus nidulans and secretion of mature interleukin-6. Bio/technology 9:378-381.
17. Zhu J, Contreras R, Fiers W. 1986. Construction of stable laboratory and industrial yeast strains expressing a foreign gene by integrative transformation using a dominant selection system. Gene 50:225-237.
18. Jou WM, Verhoeyen M, Devos R, Saman E, Fang R, Huylebroeck D, Fiers W, Threlfall G, Barber C, Carey N, Emtage S. 1980. Complete structure of the hemagglutinin gene from the human influenza A/Victoria/3/75 (H3N2) strain as determined from cloned DNA. Cell 19:683-696.
19. Verhoeyen M, Fang R, Jou WM, Devos R, Huylebroeck D, Saman E, Fiers W. 1980. Antigenic drift between the haemagglutinin of the Hong Kong influenza strains A/Aichi/2/68 and A/Victoria/3/75. Nature 286:771-776.
20. Fang R, Min Jou W, Huylebroeck D, Devos R, Fiers W. 1981. Complete structure of A/duck/Ukraine/63 influenza hemagglutinin gene: animal virus as progenitor of human H3 Hong Kong 1968 influenza hemagglutinin. Cell 25:315-323.
21. Neirynck S, Deroo T, Saelens X, Vanlandschoot P, Jou WM, Fiers W. 1999. A universal influenza A vaccine based on the extracellular domain of the M2 protein. Nature medicine 5:1157-1163.
22. Pendzialek J, Roose K, Smet A, Schepens B, Kufer P, Raum T, Baeuerle PA, Muenz M, Saelens X, Fiers W. 2017. Bispecific T cell engaging antibody constructs targeting a universally conserved part of the viral M2 ectodomain cure and prevent influenza A virus infection. Antiviral research 141:155-164.