Microfluidic characterisation reveals broad range of SARS-CoV-2 antibody affinity in human plasma.

Funder: Herchel Smith FundFunder: St John’s College CambridgeFunder: Centre for Misfolding Diseases, CambridgeFunder: Swiss FCS and the Forschungskredit of the University of ZurichFunder: Frances and Augustus Newman FoundationFunder: BBRSCFunder: NOMIS FoundationThe clinical outcome of SARS-CoV-2 infections, which can range from asymptomatic to lethal, is crucially shaped by the concentration of antiviral antibodies and by their affinity to their targets. However, the affinity of polyclonal antibody responses in plasma is difficult to measure. Here we used microfluidic antibody affinity profiling (MAAP) to determine the aggregate affinities and concentrations of anti-SARS-CoV-2 antibodies in plasma samples of 42 seropositive individuals, 19 of which were healthy donors, 20 displayed mild symptoms, and 3 were critically ill. We found that dissociation constants, K d, of anti-receptor-binding domain antibodies spanned 2.5 orders of magnitude from sub-nanomolar to 43 nM. Using MAAP we found that antibodies of seropositive individuals induced the dissociation of pre-formed spike-ACE2 receptor complexes, which indicates that MAAP can be adapted as a complementary receptor competition assay. By comparison with cytopathic effect-based neutralisation assays, we show that MAAP can reliably predict the cellular neutralisation ability of sera, which may be an important consideration when selecting the most effective samples for therapeutic plasmapheresis and tracking the success of vaccinations

Effects of troglitazone and other thiazolidinediones on the bloodstream form of Trypanosoma brucei - induction of slender form-differentiation

Werden metazyklische Formen des Parasiten Trypanosoma brucei durch einen Stich der Tsetse-Fliege von deren Speicheldrüsen auf das Blut eines Vertebraten übertragen, so differenzieren sie sofort in Blutform-Parasiten, die als slender-Formen bezeichnet werden. Diese besitzen eine hohe Teilungsrate und sind für den Anstieg der Parasitämie verantwortlich. Haben sie eine gewisse Zelldichte im Blut ihres Wirts erreicht, so differenzieren sie in dickere, teilungsdefiziente Zellen, die stumpy-Formen. Diese Differenzierung verläuft transient über eine intermediäre Blutform. Während ihres Infektionsverlaufes produziert die Blutform mehrere Prostaglandin-Derivate, darunter auch Prostaglandin D2. Nach der Sezernierung durch die Parasiten wird dieses im Blut schnell zu Prostaglandinen der J-Serie wie 15-desoxy-PGJ2 metabolisiert, die alle einen Programmierten Zelltod in den Parasiten induzieren. Thiazolidindione besitzen das gleiche Targetprotein (PPARgamma) wie 15-desoxy-PGJ2, aber bei wesentlich höherer Affinität. Daher wurden die Effekte von Troglitazon, Ciglitazon und Rosiglitazon untersucht, um einen möglichen Mechanismus des Prostaglandin-induzierten Zelltodes aufzudecken. Alle Thiazolidindione führten unter Kulturbedingungen zu einer konzentrationsabhängigen Wachstumshemmung, wobei Troglitazon mit einer IC50 von 42 µM den stärksten Effekt zeigte (bei einer Plasmaproteinbindungsrate von 99%). Im Gegensatz zu den Prostaglandinen verursachten die Thiazolidindione jedoch keinen Zellzyklusarrest, Verlust des mitochondrialen Membranpotentials oder DNA-Degradierung, was mittels FACS-Analyse und Elektronenmikroskopie untersucht worden war. Neben Apoptose konnte auch Nekrose durch diese Methoden ausgeschlossen werden. Die vor allem durch Troglitazon induzierten Veränderungen umfassten hingegen eine Proliferation der Glykosomen als auch eine deutliche Weiterentwicklung des Mitochondriums. Dabei konnte die Steigerung der mitochondrialen Aktivität nach Behandlung der Zellen durch verschiedene Parameter wie Steigerung des mitochondrialen Membranpotentials, erhöhte Cyanosensitivität hinsichtlich des Sauerstoff-Verbrauchs und Aktivität der Succinatdehydrogenase bestätigt werden. Zudem konnten sich mit Troglitazon behandelte Blutformen aus der logarithmischen Wachstumsphase im Vergleich zu Kontrollzellen wesentlich besser und schneller zu prozyklischen Zellen entwickeln, wenn sie einem in vitro Transformationsprotokoll unterzogen wurden. Microarray-Analysen bestätigten die Ergebnisse dieser Versuche und zeigten eine schon fortgeschrittenere Differenzierung von slender Parasiten zu intermediären Formen, die im Gegensatz zu stumpy Formen jedoch nicht im Zellzyklus aufgelaufen sind. Begleitet waren diese Veränderungen beispielsweise durch eine Hochregulation von ESAG4, das für eine Adenylatzyklase kodiert. Diese könnte für die begleitende intrazelluläre Konzentrationserhöhung des cAMP bei der Differenzierung verantwortlich sein, da sie ausschließlich in Blutformen exprimiert wird. Am auffälligsten war jedoch eine Steigerung von ESAG8-Transkripten, einem potentiellen regulatorischen Protein, das seine Wirkung wahrscheinlich durch Interaktion mit anderen Proteinen entfaltet. Basierend auf diesen Ergebnissen lässt sich der bisher sehr rudimentär geklärte Differenzierungsmechanismus der Blutformen weiter untersuchen. Gleichzeitig wird die Rolle der stumpy-Formen für die Vollendung des Lebenszyklus in Frage gestellt. Vielmehr scheint eine Differenzierung zu intermediären Formen für eine Weiterentwicklung der Parasiten im Darm der Fliege zu prozyklischen Formen ausreichend zu sein. Die Bedeutung der zellzyklusarretierten stumpy-Formen läge dann eher in einer Zelldichteregulation im Blut des Wirtes, um eine längere Infektion und so die Wahrscheinlichkeit einer Wiederaufnahme durch die Fliege zu gewährleisten.When metacyclic parasites enter the bloodstream of the vertebrate host, they differentiate immediately to slender bloodstream forms. They divide very rapidly and are responsible for the increase of the parasitemia. When they have reached a certain cell density, they change into stumpy forms which cease in proliferation. This differentiation is a transient process traversing an intermediate state already featured with the metabolism of the stumpy form, but morphologically difficult to determine. During the course of infection, the bloodstream form produces several prostaglandin-derivatives, among them prostaglandin D2. After releasing this substance into the bloodstream, it is rapidly metabolized in prostaglandins of the J-series like 15-deoxy-PGJ2, which finally induce a programmed cell death in the parasites. Thiazolidinediones share the target protein PPARgamma with 15-deoxy-PGJ2, but exhibit a much higher affinity. For this reason the effects of troglitazone, ciglitazone and rosiglitazone were used for further studies in order to discover a possible mechanism for the prostaglandin-effects. Under cultivation all thiazolidinediones lead to a concentration-dependent growth arrest. With an IC50 of 42 µM (and a plasma protein binding rate of 99%), troglitazone had the strongest effect on the cells. Nevertheless, in contrast to the prostaglandins, FACS-analyses and electron microscopy of thiazolidinedione-treated cells showed no sign of a cell cycle arrest, loss of mitochondrial membrane potential or DNA degradation. Beside apoptosis, necrosis also could be ruled out this way. The most obvious effects induced especially by troglitazone, were a proliferation of glycosomes and a further development of the mitochondrion. The mitochondrial activation following treatment could be confirmed by FACS-analysis, showing an increase of mitochondrial membrane potential, a raising cyano-sensitivity and a higher activity of succinate dehydrogenase. Additionally, troglitazone treated cells out of the logarithmic phase exhibited an extremely high ability to transform to procyclic parasites compared to the control cells, when they were object to an in vitro transformation protocol. Microarray analyses supported the results out of the other experiments and showed a further differentiation of the slender bloodstream form to an intermediate form which only differs from the stumpy form with regard to cell cycle arrest. This changes were accompanied e.g. by an up-regulation of ESAG4-transcripts, encoding an adenylyl cyclase. This protein could be responsible for the increase of the intracellular cAMP-concentration during the differentiation process, since ESAG4 is only expressed in the bloodstream form stage. The most striking up-regulation concerned a potential regulatory protein, ESAG8, which is thought to act via interactions with other proteins due to its structure. Based on these studies, the mechanism of bloodstream form differentiation which is only rudimentary solved, could be investigated further. Simultaneously the role of stumpy-forms for life cycle progression becomes questionable. Obviously a differentiation to an intermediate stage is sufficient for a development to procyclic parasites within the midgut of the tsetse-fly. The task of the cell cycle arrested stumpy forms would then rather be in regulation of cell density within the bloodstream, in order to ensure a longer survival of the mammalian host and an increasing likelihood of the parasites’ reuptake by another tsetse fly

Antiproliferative Effect of Dihydroxyacetone on Trypanosoma brucei Bloodstream Forms: Cell Cycle Progression, Subcellular Alterations, and Cell Death▿

We evaluated the effects of dihydroxyacetone (DHA) on Trypanosoma brucei bloodstream forms. DHA is considered an energy source for many different cell types. T. brucei takes up DHA readily due to the presence of aquaglyceroporins. However, the parasite is unable to use it as a carbon source because of the absence of DHA kinase (DHAK). We could not find a homolog of the relevant gene in the genomic database of T. brucei and have been unable to detect DHAK activity in cell lysates of the parasite, and the parasite died quickly if DHA was the sole energy source in the medium. In addition, during trypanosome cultivation, DHA induced growth inhibition with a 50% inhibitory concentration of about 1 mM, a concentration that is completely innocuous to mammals. DHA caused cell cycle arrest in the G2/M phase of up to 70% at a concentration of 2 mM. Also, DHA-treated parasites showed profound ultrastructural alterations, including an increase of vesicular structures within the cytosol and the presence of multivesicular bodies, myelin-like structures, and autophagy-like vacuoles, as well as a marked disorder of the characteristic mitochondrion structure. Based on the toxicity of DHA for trypanosomes compared with mammals, we consider DHA a starting point for a rational design of new trypanocidal drugs

Microfluidic characterisation reveals broad range of SARS-CoV-2 antibody affinity in human plasma

The clinical outcome of SARS-CoV-2 infections, which can range from asymptomatic to lethal, is crucially shaped by the concentration of antiviral antibodies and by their affinity to their targets. However, the affinity of polyclonal antibody responses in plasma is difficult to measure. Here we used microfluidic antibody affinity profiling (MAAP) to determine the aggregate affinities and concentrations of anti–SARS-CoV-2 antibodies in plasma samples of 42 seropositive individuals, 19 of which were healthy donors, 20 displayed mild symptoms, and 3 were critically ill. We found that dissociation constants, Kd, of anti–receptor-binding domain antibodies spanned 2.5 orders of magnitude from sub-nanomolar to 43 nM. Using MAAP we found that antibodies of seropositive individuals induced the dissociation of pre-formed spike-ACE2 receptor complexes, which indicates that MAAP can be adapted as a complementary receptor competition assay. By comparison with cytopathic effect–based neutralisation assays, we show that MAAP can reliably predict the cellular neutralisation ability of sera, which may be an important consideration when selecting the most effective samples for therapeutic plasmapheresis and tracking the success of vaccinations

Antibody Affinity Governs the Inhibition of SARS-CoV-2 Spike/ACE2 Binding in Patient Serum

The humoral immune response plays a key role in suppressing the pathogenesis of SARS-CoV-2. The molecular determinants underlying the neutralization of the virus remain, however, incompletely understood. Here, we show that the ability of antibodies to disrupt the binding of the viral spike protein to the angiotensin-converting enzyme 2 (ACE2) receptor on the cell, the key molecular event initiating SARS-CoV-2 entry into host cells, is controlled by the affinity of these antibodies to the viral antigen. By using microfluidic antibody-affinity profiling, we were able to quantify the serum-antibody mediated inhibition of ACE2–spike binding in two SARS-CoV-2 seropositive individuals. Measurements to determine the affinity, concentration, and neutralization potential of antibodies were performed directly in human serum. Using this approach, we demonstrate that the level of inhibition in both samples can be quantitatively described using the dissociation constants (KD) of the binary interactions between the ACE2 receptor and the spike protein as well as the spike protein and the neutralizing antibody. These experiments represent a new type of in-solution receptor binding competition assay, which has further potential applications, ranging from decisions on donor selection for convalescent plasma therapy, to identification of lead candidates in therapeutic antibody development, and vaccine development

Microfluidic Antibody Affinity Profiling Reveals the Role of Memory Reactivation and Cross-Reactivity in the Defense Against SARS-CoV-2.

Funder: Universit?tsspital Z?richFunder: Biotechnology and Biological Sciences Research CouncilFunder: NOMIS StiftungFunder: Universit?t Z?richRecent efforts in understanding the course and severity of SARS-CoV-2 infections have highlighted both potentially beneficial and detrimental effects of cross-reactive antibodies derived from memory immunity. Specifically, due to a significant degree of sequence similarity between SARS-CoV-2 and other members of the coronavirus family, memory B-cells that emerged from previous infections with endemic human coronaviruses (HCoVs) could be reactivated upon encountering the newly emerged SARS-CoV-2, thus prompting the production of cross-reactive antibodies. Determining the affinity and concentration of these potentially cross-reactive antibodies to the new SARS-CoV-2 antigens is therefore particularly important when assessing both existing immunity against common HCoVs and adverse effects like antibody-dependent enhancement (ADE) in COVID-19. However, these two fundamental parameters cannot easily be disentangled by surface-based assays like enzyme-linked immunosorbent assays (ELISAs), which are routinely used to assess cross-reactivity. Here, we have used microfluidic antibody affinity profiling (MAAP) to quantitatively evaluate the humoral immune response in COVID-19 convalescent patients by determining both antibody affinity and concentration against spike antigens of SARS-CoV-2 directly in nine convalescent COVID-19 patient and three pre-pandemic sera that were seropositive for common HCoVs. All 12 sera contained low concentrations of high-affinity antibodies against spike antigens of HCoV-NL63 and HCoV-HKU1, indicative of past exposure to these pathogens, while the affinity against the SARS-CoV-2 spike protein was lower. These results suggest that cross-reactivity as a consequence of memory reactivation upon an acute SARS-CoV-2 infection may not be a significant factor in generating immunity against SARS-CoV-2