Alpha-1 antitrypsin deficiency and recombinant protein sources with focus on plant sources: Updates, challenges and perspectives.

Alpha-1 antitrypsin deficiency (A1ATD) is an autosomal recessive disease characterized by low plasma levels of A1AT, a serine protease inhibitor representing the most abundant circulating antiprotease normally present at plasma levels of 1-2 g/L. The dominant clinical manifestations include predispositions to early onset emphysema due to protease/antiprotease imbalance in distal lung parenchyma and liver disease largely due to unsecreted polymerized accumulations of misfolded mutant A1AT within the endoplasmic reticulum of hepatocytes. Since 1987, the only FDA licensed specific therapy for the emphysema component has been infusions of A1AT purified from pooled human plasma at the 2020 cost of up to US $200,000/year with the risk of intermittent shortages. In the past three decades various, potentially less expensive, recombinant forms of human A1AT have reached early stages of development, one of which is just reaching the stage of human clinical trials. The focus of this review is to update strategies for the treatment of the pulmonary component of A1ATD with some focus on perspectives for therapeutic production and regulatory approval of a recombinant product from plants. We review other competitive technologies for treating the lung disease manifestations of A1ATD, highlight strategies for the generation of data potentially helpful for securing FDA Investigational New Drug (IND) approval and present challenges in the selection of clinical trial strategies required for FDA licensing of a New Drug Approval (NDA) for this disease.

Investigation of the immunogenicity of Zika glycan loop.

BACKGROUND: Zika virus (ZIKV) is a major human pathogen and member of the Flavivirus genus. Previous studies have identified neutralizing antibodies from Zika patients that bind to quaternary epitopes across neighboring envelope (E) proteins, called E dimer epitopes (EDE). An asparagine-linked glycan on the "glycan loop" (GL) of the ZIKV envelope protein protects the functionally important "fusion loop" on the opposite E subunit in the dimer, and EDE antibodies have been shown to bind to both of these loops. Human EDE antibodies have been divided into two subclasses based on how they bind to the glycan loop region: EDE1 antibodies do not require glycosylation for binding, while EDE2 antibodies strongly rely on the glycan for binding. METHODS: ZIKV GL was expressed on tobacco mosaic virus nanoparticles. Mice were immunized with GL or full-length monomeric E and the immune response was analyzed by testing the ability of sera and monoclonal antibodies to bind to GL and to neutralize ZIKV in in vitro cellular assay. RESULTS: We report here the existence of ZIKV moderately neutralizing antibodies that bind to E monomers through epitopes that include the glycan loop. We show that sera from human Zika patients contain antibodies capable of binding to the unglycosylated glycan loop in the absence of the rest of the envelope protein. Furthermore, mice were inoculated with recombinant E monomers and produced neutralizing antibodies that either recognize unglycosylated glycan loop or require glycan for their binding to monomeric E. We demonstrate that both types of antibodies neutralize ZIKV to some extent in a cellular virus neutralization assay. CONCLUSIONS: Analogous to the existing EDE antibody nomenclature, we propose a new classification for antibodies that bind to E monomer epitopes (EME): EME1 and EME2 for those that do not require and those that do require glycan for binding to E, respectively.

Structural Control of Nonnative Ligand Binding in Engineered Mutants of Phosphoenolpyruvate Carboxykinase.

Protein engineering to alter recognition underlying ligand binding and activity has enormous potential. Here, ligand binding for Escherichia coli phosphoenolpyruvate carboxykinase (PEPCK), which converts oxaloacetate into CO2 and phosphoenolpyruvate as the first committed step in gluconeogenesis, was engineered to accommodate alternative ligands as an exemplary system with structural information. From our identification of bicarbonate binding in the PEPCK active site at the supposed CO2 binding site, we probed binding of nonnative ligands with three oxygen atoms arranged to resemble the bicarbonate geometry. Crystal structures of PEPCK and point mutants with bound nonnative ligands thiosulfate and methanesulfonate along with strained ATP and reoriented oxaloacetate intermediates and unexpected bicarbonate were determined and analyzed. The mutations successfully altered the bound ligand position and orientation and its specificity: mutated PEPCKs bound either thiosulfate or methanesulfonate but never both. Computational calculations predicted a methanesulfonate binding mutant and revealed that release of the active site ordered solvent exerts a strong influence on ligand binding. Besides nonnative ligand binding, one mutant altered the Mn2+ coordination sphere: instead of the canonical octahedral ligand arrangement, the mutant in question had an only five-coordinate arrangement. From this work, critical features of ligand binding, position, and metal ion cofactor geometry required for all downstream events can be engineered with small numbers of mutations to provide insights into fundamental underpinnings of protein-ligand recognition. Through structural and computational knowledge, the combination of designed and random mutations aids in the robust design of predetermined changes to ligand binding and activity to engineer protein function.

Novel plant-derived recombinant human interferons with broad spectrum antiviral activity.

Type I interferons (IFNs) are potent mediators of the innate immune response to viral infection. IFNs released from infected cells bind to a receptor (IFNAR) on neighboring cells, triggering signaling cascades that limit further infection. Subtle variations in amino acids can alter IFNAR binding and signaling outcomes. We used a new gene crossbreeding method to generate hybrid, type I human IFNs with enhanced antiviral activity against four dissimilar, highly pathogenic viruses. Approximately 1400 novel IFN genes were expressed in plants, and the resultant IFN proteins were screened for antiviral activity. Comparing the gene sequences of a final set of 12 potent IFNs to those of parent genes revealed strong selection pressures at numerous amino acids. Using three-dimensional models based on a recently solved experimental structure of IFN bound to IFNAR, we show that many but not all of the amino acids that were highly selected for are predicted to improve receptor binding.

Chimeras between oilseed rape mosaic virus and tobacco mosaic virus highlight the relevant role of the tobamoviral RdRp as pathogenicity determinant in several hosts.

Oilseed rape mosaic virus (ORMV) is a tobamovirus taxonomically distinct from the type member of the genus, Tobacco mosaic virus (TMV). Both viruses display a specific host range, although they share certain hosts, such as Arabidopsis thaliana, Nicotiana benthamiana and N. tabacum, on which they induce different symptoms. Using a gain-of-symptom approach, we generated chimeric viruses, starting from a TMV infectious clone, over which different regions of ORMV were exchanged with their corresponding regions in the TMV genome. This approach allowed the association of pathogenicity determinants to certain genes within the ORMV genome. A general trend was observed associating the viral origin of the RNA-dependent RNA-polymerase (RdRp) gene and the gain of symptoms. In A. thaliana and N. benthamiana, chimeric viruses were unable to reproduce the symptoms induced by the parental viruses, leading to disease states which could be described as intermediate, and variable in some cases. In contrast, a hypersensitive reaction caused by both of these viruses on N-gene-bearing tobaccos could be found in resistance reactions to all chimeric viruses, suggesting that the avirulence determinant maps similarly in both viruses. A systemic necrotic spotting typical of non-N-gene tobaccos infected with ORMV was associated with the polymerase domain of RdRp. To our knowledge, this is the first time that this controversial portion of the tobamovirus genome has been identified directly as a pathogenicity determinant. None of the reactions of the chimeric viruses could be correlated with increases or decreases in virus titres in the infections.

Analysis of local and systemic spread of the crucifer-infecting TMV-Cg virus in tobacco and several Arabidopsis thaliana ecotypes.

The crucifer-infecting tobacco mosaic virus, TMV-Cg, infects Arabidopsis thaliana (L.) Heynh. efficiently without causing severe symptoms. The systemic spread of TMV-Cg in Arabidopsis was evaluated in 14ecotypes. Five days after inoculation, TMV-Cg was detected in apical leaves of 8 out of 14 ecotypes. As expected, the spread of TMV-Cg in the ecotypes tested was considerably faster than that of tobacco mosaic virus (TMV-U1). To study the participation of viral proteins in the TMV-Cg-induced infection, a complete genomic cDNA of TMV-Cg was cloned. The role of TMV-Cg movement protein in systemic spread was tested with a hybrid virus, constructed from the TMV-U1 genome and the TMV-Cg movement protein gene. Contrary to expectations, the systemic spread of this hybrid in Arabidopsis was similar to that of TMV-U1. The failure of the hybrid virus to spread at rates similar to those of TMV-Cg was not due to restrictions in local movement. In tobacco (Nicotiana tabacum L.), the hybrid virus spread efficiently and induced systemic mosaic symptoms characteristic of TMV-U1. The TMV-Cg cDNA clone provides an attractive tool to study virus-host interactions.

Epigenetic modification of plants with systemic RNA viruses.

Knowledge of gene function is critical to the development of new plant traits for improved agricultural and industrial applications. Viral expression vectors offer a rapid and proven method to provide epigenetic expression of foreign sequences throughout infected plants. Expression of these sequences from viral vectors can lead to gain- or loss-of-function phenotypes, allowing gene function to be determined by phenotypic or biochemical effects in the infected plant. Tobacco mosaic virus and barley stripe mosaic virus expression vectors have been developed to express foreign gene sequences in dicotyledonous and monocotyledonous hosts, respectively. Large-scale application of both viral vector systems for gene function discovery in Nicotiana and barley hosts resulted in high infection rates and produced distinctive visual phenotypes in approximately 5% of transfected plants. Novel genes expressing potential herbicide target proteins in addition to genes promoting stem elongation, leaf development and apical dominance were identified in the large-scale screening. This report illustrates the adaptability of viral vectors for gene function discovery in higher plants.