Successful treatment of post-influenza pseudomembranous necrotising bronchial aspergillosis with liposomal amphotericin, inhaled amphotericin B, gamma interferon and GM-CSF

A case of aspergillus tracheobronchitis following influenza A infection in an immunocompetent 35 year old woman is described that required prolonged mechanical ventilation for airways obstruction. Treatment included liposomal amphotericin, inhaled amphotericin, gamma interferon and GM-CSF. Liposomal amphotericin therapy was associated with reversible hepatosplenomegaly. Inhaled corticosteroids with continued antifungal therapy were used for the management of severe recurrent airway obstruction. After a prolonged course of treatment she survived with fixed airways obstruction unresponsive to corticosteroids.

Newly discovered viruses of flying foxes

Flying foxes have been the focus of research into three newly described viruses from the order Mononegavirales, namely Hendra virus (HeV), Menangle virus and Australian Bat Lyssavirus (ABL). Early investigations indicate that flying foxes are the reservoir host for these viruses. In 1994, two outbreaks of a new zoonotic disease affecting horses and humans occurred in Queensland. The virus which was found to be responsible was called equine morbillivirus (EMV) and has since been renamed HeV. Investigation into the reservoir of HeV has produced evidence that antibodies capable of neutralising HeV have only been detected in flying foxes. Over 20% of flying foxes in eastern Australia have been identified as being seropositive. Additionally six species of flying foxes in Papua New Guinea have tested positive for antibodies to HeV. In 1996 a virus from the family Paramyxoviridae was isolated from the uterine fluid of a female flying fox. Sequencing of 10 000 of the 18 000 base pairs (bp) has shown that the sequence is identical to the HeV sequence. As part of investigations into HeV, a virus was isolated from a juvenile flying fox which presented with neurological signs in 1996. This virus was characterised as belonging to the family Rhabdoviridae, and was named ABL. Since then four flying fox species and one insectivorous species have tested positive for ABL. The third virus to be detected in flying foxes is Menangle virus, belonging to the family Paramyxoviridae. This virus was responsible for a zoonotic disease affecting pigs and humans in New South Wales in 1997. Antibodies capable of neutralising Menangle virus, were detected in flying foxes. (C) 1999 Elsevier Science B.V. All rights reserved.

Viruses and the eye

About 60% of human infections diseases are caused by viruses,including such important diseases as AIDS, polio, rabies and certain forms of cancer. A few groups of viruses are important to optometrists because they either cause a primary eye infection or a systemic viral infection with ocular complications.

Editorial overview:new protein production tools for structural biology

Full text: The idea of producing proteins from recombinant DNA hatched almost half a century ago. In his PhD thesis, Peter Lobban foresaw the prospect of inserting foreign DNA (from any source, including mammalian cells) into the genome of a λ phage in order to detect and recover protein products from Escherichia coli [ 1 and 2]. Only a few years later, in 1977, Herbert Boyer and his colleagues succeeded in the first ever expression of a peptide-coding gene in E. coli — they produced recombinant somatostatin [ 3] followed shortly after by human insulin. The field has advanced enormously since those early days and today recombinant proteins have become indispensable in advancing research and development in all fields of the life sciences. Structural biology, in particular, has benefitted tremendously from recombinant protein biotechnology, and an overwhelming proportion of the entries in the Protein Data Bank (PDB) are based on heterologously expressed proteins. Nonetheless, synthesizing, purifying and stabilizing recombinant proteins can still be thoroughly challenging. For example, the soluble proteome is organized to a large part into multicomponent complexes (in humans often comprising ten or more subunits), posing critical challenges for recombinant production. A third of all proteins in cells are located in the membrane, and pose special challenges that require a more bespoke approach. Recent advances may now mean that even these most recalcitrant of proteins could become tenable structural biology targets on a more routine basis. In this special issue, we examine progress in key areas that suggests this is indeed the case. Our first contribution examines the importance of understanding quality control in the host cell during recombinant protein production, and pays particular attention to the synthesis of recombinant membrane proteins. A major challenge faced by any host cell factory is the balance it must strike between its own requirements for growth and the fact that its cellular machinery has essentially been hijacked by an expression construct. In this context, Bill and von der Haar examine emerging insights into the role of the dependent pathways of translation and protein folding in defining high-yielding recombinant membrane protein production experiments for the common prokaryotic and eukaryotic expression hosts. Rather than acting as isolated entities, many membrane proteins form complexes to carry out their functions. To understand their biological mechanisms, it is essential to study the molecular structure of the intact membrane protein assemblies. Recombinant production of membrane protein complexes is still a formidable, at times insurmountable, challenge. In these cases, extraction from natural sources is the only option to prepare samples for structural and functional studies. Zorman and co-workers, in our second contribution, provide an overview of recent advances in the production of multi-subunit membrane protein complexes and highlight recent achievements in membrane protein structural research brought about by state-of-the-art near-atomic resolution cryo-electron microscopy techniques. E. coli has been the dominant host cell for recombinant protein production. Nonetheless, eukaryotic expression systems, including yeasts, insect cells and mammalian cells, are increasingly gaining prominence in the field. The yeast species Pichia pastoris, is a well-established recombinant expression system for a number of applications, including the production of a range of different membrane proteins. Byrne reviews high-resolution structures that have been determined using this methylotroph as an expression host. Although it is not yet clear why P. pastoris is suited to producing such a wide range of membrane proteins, its ease of use and the availability of diverse tools that can be readily implemented in standard bioscience laboratories mean that it is likely to become an increasingly popular option in structural biology pipelines. The contribution by Columbus concludes the membrane protein section of this volume. In her overview of post-expression strategies, Columbus surveys the four most common biochemical approaches for the structural investigation of membrane proteins. Limited proteolysis has successfully aided structure determination of membrane proteins in many cases. Deglycosylation of membrane proteins following production and purification analysis has also facilitated membrane protein structure analysis. Moreover, chemical modifications, such as lysine methylation and cysteine alkylation, have proven their worth to facilitate crystallization of membrane proteins, as well as NMR investigations of membrane protein conformational sampling. Together these approaches have greatly facilitated the structure determination of more than 40 membrane proteins to date. It may be an advantage to produce a target protein in mammalian cells, especially if authentic post-translational modifications such as glycosylation are required for proper activity. Chinese Hamster Ovary (CHO) cells and Human Embryonic Kidney (HEK) 293 cell lines have emerged as excellent hosts for heterologous production. The generation of stable cell-lines is often an aspiration for synthesizing proteins expressed in mammalian cells, in particular if high volumetric yields are to be achieved. In his report, Buessow surveys recent structures of proteins produced using stable mammalian cells and summarizes both well-established and novel approaches to facilitate stable cell-line generation for structural biology applications. The ambition of many biologists is to observe a protein's structure in the native environment of the cell itself. Until recently, this seemed to be more of a dream than a reality. Advances in nuclear magnetic resonance (NMR) spectroscopy techniques, however, have now made possible the observation of mechanistic events at the molecular level of protein structure. Smith and colleagues, in an exciting contribution, review emerging ‘in-cell NMR’ techniques that demonstrate the potential to monitor biological activities by NMR in real time in native physiological environments. A current drawback of NMR as a structure determination tool derives from size limitations of the molecule under investigation and the structures of large proteins and their complexes are therefore typically intractable by NMR. A solution to this challenge is the use of selective isotope labeling of the target protein, which results in a marked reduction of the complexity of NMR spectra and allows dynamic processes even in very large proteins and even ribosomes to be investigated. Kerfah and co-workers introduce methyl-specific isotopic labeling as a molecular tool-box, and review its applications to the solution NMR analysis of large proteins. Tyagi and Lemke next examine single-molecule FRET and crosslinking following the co-translational incorporation of non-canonical amino acids (ncAAs); the goal here is to move beyond static snap-shots of proteins and their complexes and to observe them as dynamic entities. The encoding of ncAAs through codon-suppression technology allows biomolecules to be investigated with diverse structural biology methods. In their article, Tyagi and Lemke discuss these approaches and speculate on the design of improved host organisms for ‘integrative structural biology research’. Our volume concludes with two contributions that resolve particular bottlenecks in the protein structure determination pipeline. The contribution by Crepin and co-workers introduces the concept of polyproteins in contemporary structural biology. Polyproteins are widespread in nature. They represent long polypeptide chains in which individual smaller proteins with different biological function are covalently linked together. Highly specific proteases then tailor the polyprotein into its constituent proteins. Many viruses use polyproteins as a means of organizing their proteome. The concept of polyproteins has now been exploited successfully to produce hitherto inaccessible recombinant protein complexes. For instance, by means of a self-processing synthetic polyprotein, the influenza polymerase, a high-value drug target that had remained elusive for decades, has been produced, and its high-resolution structure determined. In the contribution by Desmyter and co-workers, a further, often imposing, bottleneck in high-resolution protein structure determination is addressed: The requirement to form stable three-dimensional crystal lattices that diffract incident X-ray radiation to high resolution. Nanobodies have proven to be uniquely useful as crystallization chaperones, to coax challenging targets into suitable crystal lattices. Desmyter and co-workers review the generation of nanobodies by immunization, and highlight the application of this powerful technology to the crystallography of important protein specimens including G protein-coupled receptors (GPCRs). Recombinant protein production has come a long way since Peter Lobban's hypothesis in the late 1960s, with recombinant proteins now a dominant force in structural biology. The contributions in this volume showcase an impressive array of inventive approaches that are being developed and implemented, ever increasing the scope of recombinant technology to facilitate the determination of elusive protein structures. Powerful new methods from synthetic biology are further accelerating progress. Structure determination is now reaching into the living cell with the ultimate goal of observing functional molecular architectures in action in their native physiological environment. We anticipate that even the most challenging protein assemblies will be tackled by recombinant technology in the near future.

Towards the knowledge-based design of universal influenza epitope ensemble vaccines

Motivation: Influenza A viral heterogeneity remains a significant threat due to unpredictable antigenic drift in seasonal influenza and antigenic shifts caused by the emergence of novel subtypes. Annual review of multivalent influenza vaccines targets strains of influenza A and B likely to be predominant in future influenza seasons. This does not induce broad, cross protective immunity against emergent subtypes. Better strategies are needed to prevent future pandemics. Cross-protection can be achieved by activating CD8+ and CD4+ T cells against highly-conserved regions of the influenza genome. We combine available experimental data with informatics-based immunological predictions to help design vaccines potentially able to induce cross-protective T-cells against multiple influenza subtypes. Results: To exemplify our approach we designed two epitope ensemble vaccines comprising highly-conserved and experimentally-verified immunogenic influenza A epitopes as putative non-seasonal influenza vaccines; one specifically targets the US population and the other is a universal vaccine. The USA-specific vaccine comprised 6 CD8+ T cell epitopes (GILGFVFTL, FMYSDFHFI, GMDPRMCSL, SVKEKDMTK, FYIQMCTEL, DTVNRTHQY) and 3 CD4+ epitopes (KGILGFVFTLTVPSE, EYIMKGVYINTALLN, ILGFVFTLTVPSERG). The universal vaccine comprised 8 CD8+ epitopes: (FMYSDFHFI, GILGFVFTL, ILRGSVAHK, FYIQMCTEL, ILKGKFQTA, YYLEKANKI, VSDGGPNLY, YSHGTGTGY) and the same 3 CD4+ epitopes. Our USA-specific vaccine has a population protection coverage (portion of the population potentially responsive to one or more component epitopes of the vaccine, PPC) of over 96% and 95% coverage of observed influenza subtypes. The universal vaccine has a PPC value of over 97% and 88% coverage of observed subtypes.