A scalable and reproducible manufacturing process for Phlebotomus papatasi salivary protein PpSP15, a vaccine candidate for leishmaniasis.

Cutaneous leishmaniasis is a parasitic and neglected tropical disease transmitted by the bites of sandflies. The emergence of cutaneous leishmaniasis in areas of war, conflict, political instability, and climate change has prompted efforts to develop a preventive vaccine. One vaccine candidate antigen is PpSP15, a 15 kDa salivary antigen from the sandfly Phlebotomus papatasi that facilitates the infection of the Leishmania parasite and has been shown to induce parasite-specific cell-mediated immunity. Previously, we developed a fermentation process for producing recombinant PpSP15 in Pichia pastoris and a two-chromatographic-step purification process at 100 mL scale. Here we expand the process design to the 10 L scale and examine its reproducibility by performing three identical process runs, an essential transition step towards technology transfer for pilot manufacture. The process was able to reproducibly recover 81% of PpSP15 recombinant protein with a yield of 0.75 g/L of fermentation supernatant, a purity level of 97% and with low variance among runs. Additionally, a freeze-thaw stability study indicated that the PpSP15 recombinant protein remains stable after undergoing three freeze-thaw cycles, and an accelerated stability study confirmed its stability at 37 °C for at least one month. A research cell bank for the expression of PpSP15 was generated and fully characterized. Collectively, the cell bank and the production process are ready for technology transfer for future cGMP pilot manufacturing.

Therapeutic target-site variability in α1-antitrypsin characterized at high resolution.

The intrinsic propensity of α(1)-antitrypsin to undergo conformational transitions from its metastable native state to hyperstable forms provides a motive force for its antiprotease function. However, aberrant conformational change can also occur via an intermolecular linkage that results in polymerization. This has both loss-of-function and gain-of-function effects that lead to deficiency of the protein in human circulation, emphysema and hepatic cirrhosis. One of the most promising therapeutic strategies being developed to treat this disease targets small molecules to an allosteric site in the α(1)-antitrypsin molecule. Partial filling of this site impedes polymerization without abolishing function. Drug development can be improved by optimizing data on the structure and dynamics of this site. A new 1.8 Å resolution structure of α(1)-antitrypsin demonstrates structural variability within this site, with associated fluctuations in its upper and lower entrance grooves and ligand-binding characteristics around the innermost stable enclosed hydrophobic recess. These data will allow a broader selection of chemotypes and derivatives to be tested in silico and in vitro when screening and developing compounds to modulate conformational change to block the pathological mechanism while preserving function.

Process Characterization and Biophysical Analysis for a Yeast-Expressed Phlebotomus papatasi Salivary Protein (PpSP15) as a Leishmania Vaccine Candidate.

Cutaneous leishmaniasis is a neglected tropical disease caused by the parasite Leishmania and transmitted by sandflies. It has become a major health problem in many tropical and subtropical countries, especially in regions of conflict and political instability. Currently, there are only limited drug treatments and no available licensed vaccine; thus, the need for more therapeutic interventions remains urgent. Previously, a DNA vaccine encoding a 15 kDa sandfly (Phlebotomus papatasi) salivary protein (PpSP15) and recombinant nonpathogenic Leishmania tarentolae secreting PpSP15 have been shown to induce protective immunity against Leishmania major in mice, demonstrating that PpSP15 is a promising vaccine candidate. In this study, we developed a fermentation process in yeast with a yield of ~1g PpSP15/L and a scalable purification process consisting of only 2 chromatographic purification steps with high binding capacity for PpSP15, suggesting that PpSP15 can be produced economically. The biophysical/biochemical analysis of the purified PpSP15 indicated that the protein was of high purity (>97%) and conformationally stable between pH 4.4 and 9.0. More importantly, the recombinant protein had a defined structure similar to that of the related PdSP15 from Phlebotomus duboscqi, implying the suitability of the yeast expression system for producing a correctly folded PpSP15.

Vitamin D (1,25(OH)2D3) induces α-1-antitrypsin synthesis by CD4+ T cells, which is required for 1,25(OH)2D3-driven IL-10.

Studies to identify novel immune-regulatory functions of active vitamin D (1,25(OH)2D3) in human CD4+ T cells revealed that 1,25(OH)2D3 potently induced expression of the gene SERPINA1, encoding the anti-protease α-1-antitrypsin. We confirmed α-1-antitrypsin protein expression by 1,25(OH)2D3-treated CD4+ T cells, but not in CD8+ T cells or monocytes. α-1-Antitrypsin promotes anti-inflammatory IL-10 synthesis in other immune cell populations. We therefore investigated its immune-regulatory effects in CD4+ T cells. Plasma-derived α-1-antitrypsin drove IL-10 synthesis by CD4+ T cells, which was not dependent on anti-protease activity, but appeared to require a serum-binding factor, since this could not be achieved with recombinant protein. α-1-Antitrypsin is reported to bind complement components, which regulate T cell function. A role for this interaction was therefore probed. Plasma-derived, but not recombinant α-1-antitrypsin contained C3a. Surface Plasmon Resonance and Microscale Thermophoresis demonstrated α-1-antitrypsin binding to C3a. Addition of C3a to CD4+ T cells cultured with recombinant α-1-antitrypsin restored induction of IL-10, whereas neutralisation of C3a abrogated IL-10 induced by plasma-derived α-1-antitrypsin. To interrogate an endogenous role for the α-1-antitrypsin-C3a axis in 1,25(OH)2D3-driven CD4+ T cell IL-10 synthesis, we treated cells from healthy or α-1-antitrypsin-deficient individuals (which transcribe SERPINA1 but do not secrete protein) with 1,25(OH)2D3. A significant correlation was identified between SERPINA1 and IL10 gene expression in healthy donor CD4+ T cells, which was absent in cells from α-1-antitrypsin-deficient individuals. Therefore, α-1-antitrypsin is required for 1,25(OH)2D3-induced IL-10 expression in CD4+ T cells, interacting with C3a to drive IL-10 expression.

Crystallization and preliminary X-ray analysis of recombinant Glomerella cingulata cutinase.

Cutinase catalyzes the hydrolysis of water-soluble esters and long-chain triglycerides and belongs to the family of serine hydrolases. The enzyme is thought to represent an evolutionary link between the esterase and lipase families and has potential applications in a wide range of industrial hydrolytic processes, for which an understanding of the molecular basis of its substrate specificity is critical. Glomerella cingulata cutinase has been cloned and the protein has been overexpressed in Escherichia coli, purified and subsequently crystallized in a wide range of different crystal forms in the presence and absence of inhibitors. The best crystals are those of the apo cutinase, which diffract to beyond 1.6 A resolution and belong to space group P4(1)2(1)2 or P4(3)2(1)2. Crystals of cutinase with the inhibitors PETFP or E600 belong to space groups P2(1)2(1)2(1) and P2(1), respectively, and diffract to approximately 2.5 A resolution. All of the crystals are suitable for structural studies, which are currently ongoing.

Engineering a stable CHO cell line for the expression of a MERS-coronavirus vaccine antigen.

Middle East respiratory syndrome coronavirus (MERS-CoV) has infected at least 2040 patients and caused 712 deaths since its first appearance in 2012, yet neither pathogen-specific therapeutics nor approved vaccines are available. To address this need, we are developing a subunit recombinant protein vaccine comprising residues 377-588 of the MERS-CoV spike protein receptor-binding domain (RBD), which, when formulated with the AddaVax adjuvant, it induces a significant neutralizing antibody response and protection against MERS-CoV challenge in vaccinated animals. To prepare for the manufacture and first-in-human testing of the vaccine, we have developed a process to stably produce the recombinant MERS S377-588 protein in Chinese hamster ovary (CHO) cells. To accomplish this, we transfected an adherent dihydrofolate reductase-deficient CHO cell line (adCHO) with a plasmid encoding S377-588 fused with the human IgG Fc fragment (S377-588-Fc). We then demonstrated the interleukin-2 signal peptide-directed secretion of the recombinant protein into extracellular milieu. Using a gradually increasing methotrexate (MTX) concentration to 5 µM, we increased protein yield by a factor of 40. The adCHO-expressed S377-588-Fc recombinant protein demonstrated functionality and binding specificity identical to those of the protein from transiently transfected HEK293T cells. In addition, hCD26/dipeptidyl peptidase-4 (DPP4) transgenic mice vaccinated with AddaVax-adjuvanted S377-588-Fc could produce neutralizing antibodies against MERS-CoV and survived for at least 21 days after challenge with live MERS-CoV with no evidence of immunological toxicity or eosinophilic immune enhancement. To prepare for large scale-manufacture of the vaccine antigen, we have further developed a high-yield monoclonal suspension CHO cell line.

Vaccine Development Against Middle East Respiratory Syndrome.

Various types of vaccines are under pre-clinical and clinical development to address the recent appearance of Middle East respiratory syndrome or MERS, an emerging infectious disease that has already caused over 600 deaths and remains a threat to world health. The causative agent for this respiratory disease is a member of the betacoronavirus genus, phylogenetically closely related to the SARS coronavirus that caused an international health emergency in 2002. With lessons learned from the outbreak of severe acute respiratory syndrome, and with undeniable technological advances, vaccine development against MERS was initially fast-paced and has produced several DNA and protein vaccine candidates with promising results during early pre-clinical testing. At least one vaccine candidate has even entered first-in-humans clinical trials now. With the number of MERS cases declining though and other infectious diseases attracting increased attention, the question remains, whether, similar to the situation after the SARS pandemic, vaccine development is halted or remains the priority it rightfully should.

An integrative approach combining ion mobility mass spectrometry, X-ray crystallography, and nuclear magnetic resonance spectroscopy to study the conformational dynamics of α1 -antitrypsin upon ligand binding.

Native mass spectrometry (MS) methods permit the study of multiple protein species within solution equilibria, whereas ion mobility (IM)-MS can report on conformational behavior of specific states. We used IM-MS to study a conformationally labile protein (α1 -antitrypsin) that undergoes pathological polymerization in the context of point mutations. The folded, native state of the Z-variant remains highly polymerogenic in physiological conditions despite only minor thermodynamic destabilization relative to the wild-type variant. Various data implicate kinetic instability (conformational lability within a native state ensemble) as the basis of Z α1 -antitrypsin polymerogenicity. We show the ability of IM-MS to track such disease-relevant conformational behavior in detail by studying the effects of peptide binding on α1 -antitrypsin conformation and dynamics. IM-MS is, therefore, an ideal platform for the screening of compounds that result in therapeutically beneficial kinetic stabilization of native α1 -antitrypsin. Our findings are confirmed with high-resolution X-ray crystallographic and nuclear magnetic resonance spectroscopic studies of the same event, which together dissect structural changes from dynamic effects caused by peptide binding at a residue-specific level. IM-MS methods, therefore, have great potential for further study of biologically relevant thermodynamic and kinetic instability of proteins and provide rapid and multidimensional characterization of ligand interactions of therapeutic interest.

Therapeutic targeting of misfolding and conformational change in α1-antitrypsin deficiency.

Misfolding and conformational diseases are increasing in prominence and prevalence. Both misfolding and 'postfolding' conformational mechanisms can contribute to pathogenesis and can coexist. The different contexts of folding and native state behavior may have implications for the development of therapeutic strategies. α1-antitrypsin deficiency illustrates how these issues can be addressed with therapeutic approaches to rescue folding, ameliorate downstream consequences of aberrant polymerization and/or maintain physiological function. Small-molecule strategies have successfully targeted structural features of the native conformer. Recent developments include the capability to follow solution behavior of α1-antitrypsin in the context of disease mutations and interactions with drug-like compounds. Moreover, preclinical studies in cells and organisms support the potential of manipulating cellular response repertoires to process misfolded and polymer states.

1H, 15N and 13C backbone resonance assignments of the archetypal serpin α1-antitrypsin.

Alpha(1)-antitrypsin is a 45-kDa (394-residue) serine protease inhibitor synthesized by hepatocytes, which is released into the circulatory system and protects the lung from the actions of neutrophil elastase via a conformational transition within a dynamic inhibitory mechanism. Relatively common point mutations subvert this transition, causing polymerisation of α(1)-antitrypsin and deficiency of the circulating protein, predisposing carriers to severe lung and liver disease. We have assigned the backbone resonances of α(1)-antitrypsin using multidimensional heteronuclear NMR spectroscopy. These assignments provide the starting point for a detailed solution state characterization of the structural properties of this highly dynamic protein via NMR methods.

Structural dynamics associated with intermediate formation in an archetypal conformational disease.

In conformational diseases, native protein conformers convert to pathological intermediates that polymerize. Structural characterization of these key intermediates is challenging. They are unstable and minimally populated in dynamic equilibria that may be perturbed by many analytical techniques. We have characterized a forme fruste deficiency variant of α(1)-antitrypsin (Lys154Asn) that forms polymers recapitulating the conformer-specific neo-epitope observed in polymers that form in vivo. Lys154Asn α(1)-antitrypsin populates an intermediate ensemble along the polymerization pathway at physiological temperatures. Nuclear magnetic resonance spectroscopy was used to report the structural and dynamic changes associated with this. Our data highlight an interaction network likely to regulate conformational change and do not support the recent contention that the disease-relevant intermediate is substantially unfolded. Conformational disease intermediates may best be defined using powerful but minimally perturbing techniques, mild disease mutants, and physiological conditions.

Targeting serpins in high-throughput and structure-based drug design.

Native, metastable serpins inherently tend to undergo stabilizing conformational transitions in mechanisms of health (e.g., enzyme inhibition) and disease (serpinopathies). This intrinsic tendency is modifiable by ligand binding, thus structure-based drug design is an attractive strategy in the serpinopathies. This can be viewed as a labor-intensive approach, and historically, its intellectual attractiveness has been tempered by relatively limited success in development of drugs reaching clinical practice. However, the increasing availability of a range of powerful experimental systems and higher-throughput techniques is causing academic and early-stage industrial pharmaceutical approaches to converge. In this review, we outline the different systems and techniques that are bridging the gap between what have traditionally been considered distinct disciplines. The individual methods are not serpin-specific. Indeed, many have only recently been applied to serpins, and thus investigators in other fields may have greater experience of their use to date. However, by presenting examples from our work and that of other investigators in the serpin field, we highlight how techniques with potential for automation and scaling can be combined to address a range of context-specific challenges in targeting the serpinopathies.

Catalysis by Glomerella cingulata cutinase requires conformational cycling between the active and inactive states of its catalytic triad.

Cutinase belongs to a group of enzymes that catalyze the hydrolysis of esters and triglycerides. Structural studies on the enzyme from Fusarium solani have revealed the presence of a classic catalytic triad that has been implicated in the enzyme's mechanism. We have solved the crystal structure of Glomerella cingulata cutinase in the absence and in the presence of the inhibitors E600 (diethyl p-nitrophenyl phosphate) and PETFP (3-phenethylthio-1,1,1-trifluoropropan-2-one) to resolutions between 2.6 and 1.9 A. Analysis of these structures reveals that the catalytic triad (Ser136, Asp191, and His204) adopts an unusual configuration with the putative essential histidine His204 swung out of the active site into a position where it is unable to participate in catalysis, with the imidazole ring 11 A away from its expected position. Solution-state NMR experiments are consistent with the disrupted configuration of the triad observed crystallographically. H204N, a site-directed mutant, was shown to be catalytically inactive, confirming the importance of this residue in the enzyme mechanism. These findings suggest that, during its catalytic cycle, cutinase undergoes a significant conformational rearrangement converting the loop bearing the histidine from an inactive conformation, in which the histidine of the triad is solvent exposed, to an active conformation, in which the triad assumes a classic configuration.