Unglycosylated Soluble SARS-CoV-2 Receptor Binding Domain (RBD) Produced in E. coli Combined with the Army Liposomal Formulation Containing QS21 (ALFQ) Elicits Neutralizing Antibodies against Mismatched Variants

The emergence of novel potentially pandemic pathogens necessitates the rapid manufacture and deployment of effective, stable, and locally manufacturable vaccines on a global scale. In this study, the ability of the Escherichia coli expression system to produce the receptor binding domain (RBD) of the SARS-CoV-2 spike protein was evaluated. The RBD of the original Wuhan-Hu1 variant and of the Alpha and Beta variants of concern (VoC) were expressed in E. coli, and their biochemical and immunological profiles were compared to RBD produced in mammalian cells. The E. coli-produced RBD variants recapitulated the structural character of mammalian-expressed RBD and bound to human angiotensin converting enzyme (ACE2) receptor and a panel of neutralizing SARS-CoV-2 monoclonal antibodies. A pilot vaccination in mice with bacterial RBDs formulated with a novel liposomal adjuvant, Army Liposomal Formulation containing QS21 (ALFQ), induced polyclonal antibodies that inhibited RBD association to ACE2 in vitro and potently neutralized homologous and heterologous SARS-CoV-2 pseudoviruses. Although all vaccines induced neutralization of the non-vaccine Delta variant, only the Beta RBD vaccine produced in E. coli and mammalian cells effectively neutralized the Omicron BA.1 pseudovirus. These outcomes warrant further exploration of E. coli as an expression platform for non-glycosylated, soluble immunogens for future rapid response to emerging pandemic pathogens

Strukturelle und biochemische Analyse von GTPases of IMmunity-associated Proteins (GIMAPs) und ihren Interaktionspartnern

GTPases of IMmunity Associated Proteins (GIMAPs) comprise a family of septin- related GTPase associated with the adaptive immune system. They have been implicated in lymphocyte development, mitochondrial DNA segregation, apoptosis and autophagy. Structural studies revealed that some GIMAPs can form GTP- dependent protein scaffolds by oligomerizing via two interfaces on the surface of membranes. Biochemical studies indicated that other members may facilitate the disassembly of these scaffolds. The aim of this doctoral thesis was to characterize the cellular role of GIMAPs by identifying their interaction partners and to carry out an extensive structural and biochemical analysis on them. Interaction partners of GIMAPs were identified by mass spectrometric analysis and were involved in functions like apoptosis, autophagy, protein folding and vesicular trafficking. In particular, GABARAPL2, an autophagy- associated protein, and GIMAP6 were identified as specific interaction partners of GIMAP7. Subsequently, the GIMAP6-GIMAP7 interaction was biochemically characterized, and it was established that GIMAP6 can down- regulate the GTPase function of GIMAP7 at equimolar concentrations. Based on homology models and biochemical analysis, it was shown that the G domain of GIMAP6 is the main determinant for GTPase inhibition, whereas the C-terminal helices contribute to some extent. Also the interaction between GABARAPL2 and GIMAP6 was biochemically and structurally characterized. GABARAPL2 interacted with GIMAP6 with nanomolar affinity, as measured in isothermal titration calorimetry (ITC) and bio-layer interferometry. Interestingly, a C- terminal 10 amino acid peptide of GIMAP6 previously implicated in the interaction did not bind to GABARAPL2 in the ITC measurement suggesting of a novel mode of interaction. The crystal structure of GABARAPL2 was solved to atomic resolution. GABARAPL2 exhibited a conserved ubiquitin superfamily fold and oligomerized in the crystal in a head to tail fashion leading to a speculation that it might form protein scaffold on the surface of autophagosomes upon induction of autophagy. Finally, a model for the function of GIMAP6, GIMAP7 and GABARAPL2 together with GIMAP2 is proposed. Obtained findings from this thesis will form a basis for many mutational and cell biological studies in order to understand the molecular function of GIMAPs.GTPases of IMmunity Associated Proteins' (GIMAPs) sind eine septin-verwandte GTPase- Familie, die mit dem angeborenen Immunsystem assoziiert ist. GIMAPs spielen eine wichtige Rolle in der Entwicklung von Lymphozyten, der Segregation mitochondrialer DNA, Apoptose sowie in der Autophagie. Strukturstudien zeigten, dass einige GIMAPs GTP-abhängige Proteingerüste bilden, in dem sie über zwei Interaktionsflächen auf der Oberfläche von Membranen assemblieren. Biochemische Studien deuten darauf hin, dass andere GIMAP Mitglieder das Proteingerüst zerlegen können. Das Ziel dieser Doktorarbeit war es, die zelluläre Funktion von GIMAPs zu charakterisieren, in dem Interaktionspartner gefunden und biochemisch und strukturell analysiert werden. Interaktionspartner von GIMAPs wurden massenspektrometrisch identifiziert und spielen Funktionen in der Apoptose, Autophagie, Proteinfaltung sowie dem Vesikeltransport. Insbesondere wurden das Autophagie- assoziierte GABARAPL2 und GIMAP6 als spezifische Interaktionspartner von GIMAP7 gefunden. Die GIMAP6-GIMAP7 Interaktion wurde biochemisch charakterisiert; es konnte gezeigt werden, dass GIMAP6 die GTPase-Funktion von GIMAP7 bei äquimolaren Verhältnissen inhibieren kann. Basierend auf Homologiemodellen und biochemischen Analysen konnte gezeigt werden, dass die GTPase- Domäne von GIMAP6 der entscheidende Faktor für die GTPase-Inhibierung ist, zu der zusätzlich C-terminale Helices beitragen. GABARPL2 interagiert mit nanomolarer Affinität mit GIMAP6, wie durch isotherme Titrationskalorimetrie (ITC) und Bioschicht-Interferenz gezeigt werden konnte. Ein C-terminales, 10 Aminosäuren langes GIMAP6 Peptid, das in vorhergehenden Studien in der Interaktion impliziert wurde, band in ITC-Messungen nicht an GABARAPL2, was auf einen neuen Interaktionsmodus hinweist. Die Kristallstruktur von GABARAPL2 wurde bei atomarer Auflösung bestimmt. Die Struktur zeigt eine konservierte Ubiquitinfaltung und eine Kopf-zu-Schwanz Oligomerisierung im Kristall. Dies könnte darauf hindeuten, dass GABARAPL2 ein Proteingerüst auf der Oberfläche von “Autophagosomen” bilden kann. Abschließend wird in dieser Doktorarbeit ein Modell für die Funktion von GIMAP6, GIMAP7, GABARAPL2 sowie GIMAP6 vorgeschlagen. Die hier gewonnenen Erkenntnisse werden als Basis für viele weitere Mutations- sowie zellbiologische Studien dienen, die die molekulare Charakterisierung von GIMAPs zum Ziel haben

Unglycosylated Soluble SARS-CoV-2 Receptor Binding Domain (RBD) Produced in <i>E. coli</i> Combined with the Army Liposomal Formulation Containing QS21 (ALFQ) Elicits Neutralizing Antibodies against Mismatched Variants

The emergence of novel potentially pandemic pathogens necessitates the rapid manufacture and deployment of effective, stable, and locally manufacturable vaccines on a global scale. In this study, the ability of the Escherichia coli expression system to produce the receptor binding domain (RBD) of the SARS-CoV-2 spike protein was evaluated. The RBD of the original Wuhan-Hu1 variant and of the Alpha and Beta variants of concern (VoC) were expressed in E. coli, and their biochemical and immunological profiles were compared to RBD produced in mammalian cells. The E. coli-produced RBD variants recapitulated the structural character of mammalian-expressed RBD and bound to human angiotensin converting enzyme (ACE2) receptor and a panel of neutralizing SARS-CoV-2 monoclonal antibodies. A pilot vaccination in mice with bacterial RBDs formulated with a novel liposomal adjuvant, Army Liposomal Formulation containing QS21 (ALFQ), induced polyclonal antibodies that inhibited RBD association to ACE2 in vitro and potently neutralized homologous and heterologous SARS-CoV-2 pseudoviruses. Although all vaccines induced neutralization of the non-vaccine Delta variant, only the Beta RBD vaccine produced in E. coli and mammalian cells effectively neutralized the Omicron BA.1 pseudovirus. These outcomes warrant further exploration of E. coli as an expression platform for non-glycosylated, soluble immunogens for future rapid response to emerging pandemic pathogens

GIMAP5 deficiency reveals a mammalian ceramide-driven longevity assurance pathway

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