Despite the burgeoning field of coronavirus disease-19 (COVID-19) research, the persistence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralising antibodies remains unclear. This study validated two high-throughput immunological methods for use as surrogate live virus neutralisation assays and employed them to examine the half-life of SARS-CoV-2 neutralising antibodies in convalescent plasma donations made by 42 repeat donors between April and September 2020. SARS-CoV-2 neutralising antibody titres decreased over time but typically remained above the methods' diagnostic cut-offs. Using this longitudinal data, the average half-life of SARS-CoV-2 neutralising antibodies was determined to be 20.4 days. SARS-CoV-2 neutralising antibody titres appear to persist in the majority of donors for several months. Whether these titres confer protection against re-infection requires further study and is of particular relevance as COVID-19 vaccines become widely available.
Analysis of convalescent plasma derived from individuals has shown that IgG3 has the most important role in binding to SARS-CoV-2 antigens; however, this has not yet been confirmed in large studies, and the link between binding and neutralization has not been confirmed. By analyzing plasma pools consisting of 247-567 individual convalescent donors, we demonstrated the binding of IgG3 and IgM to Spike-1 protein and the receptor-binding domain correlates strongly with viral neutralization in vitro. Furthermore, despite accounting for only approximately 12% of total immunoglobulin mass, collectively IgG3 and IgM account for approximately 80% of the total neutralization. This may have important implications for the development of potent therapies for COVID-19, as it indicates that hyperimmune globulins or convalescent plasma donations with high IgG3 concentrations may be a highly efficacious therapy.
Vascular Endothelial Growth Factors (VEGFs) are key players in blood and lymphatic
vessel development and homeostasis. The family consists of five members, VEGF-A,
-B, -C, -D and placenta growth factor (PLGF). They bind to three type V receptor
tyrosine kinases (RTKs): VEGF-receptor-1 (VEGFR)-1 (Flt1), VEGFR-2 (KDR/Flk1),
and VEGFR-3 (Flt4). VEGFR-2 is the major receptor responsible for angiogenic and
vasculogenic signaling by VEGFs involving cell survival, migration and mitogenesis.
VEGFRs consist of an extracellular domain (ECD) with seven immunoglobulinhomology
domains (Ig-homology domains). The ECD is responsible for ligand binding and
contributes to the dimerization process of the receptors by forming homotypic
receptor contacts. A single transmembrane helix connects the ECD to the
intracellular kinase domain. Ligand binding to VEGFR ectodomains induces
dimerization of receptor monomers followed by autophosphorylation of specific
tyrosine residues in the intracellular kinase domains. The phosphotyrosine containing
activated kinase subsequently recruits signaling proteins thereby activating distinct
cellular pathways. Here we show that the introduction of glutamic acid residues into
the transmembrane domain (TMD) of VEGFR-2 leads to dimerization and induces
conformational changes in the TMD. A subsequent rearrangement of the intracellular
kinase domains gives rise to either active or inactive receptor dimers. We also show
that the ECD of VEGFR-2 plays an essential autoinhibitory role in the absence of
ligand. Furthermore, high-resolution structural analysis of isolated wild type (wt) and
mutant TMD by NMR spectroscopy reveal TMD conformations presumably essential
for receptor activation.
In a second project, we analysed the function of the kinase insert domain (KID) and
the C-terminal domain (CD) in VEGFR-2 activation. We show that these domains
regulate VEGFR-2 activity. The KID, and particularly a canonical tyrosine residue
located at position 951 are highly relevant for kinase activation. Deletion of the CD
renders VEGFR-2 constitutively active and we thus think that the CD of VEGFR-2
maintains the receptor in the inactive state in the absence of ligand. Low resolution
structural data derived from small angle X-ray scattering (SAXS) and MALS (Multi
Angle Light Scattering) give evidence that the kinase domain of VEGFR-2 undergoes
significant conformational changes when switching from the inactive to the active
state. The activated kinase domain adopts an elongated, open conformation whereas
the inactivated kinase domain remains in a globular compact conformation with the
CD presumably blocking the catalytic site of the kinase similar to the autoinhibited
conformation previously demonstrated for the Tie-2 kinase domain. Additional
sedimentation equilibrium analytical ultracentrifugation (AUC) experiments of kinase
domain mutants demonstrated that the KID and the CD are necessary to sustain an
intrinsic dimerization propensity that potentially supports the dimerization process
induced by ligand binding to the ECD. Deletion mutants showed lower affinity forming
dimers only at higher concentration. In experiments aiming to investigate
phosphorylation kinetics of the VEGFR-2 kinase domain we finally showed that
VEGFR-2 follows a well-ordered sequence of residue-by-residue phosphorylation.
In a third project we were interested in characterizing the in vitro interaction between
TSAd and activated VEGFR-2. Y951 mediated complex formation of TSAd with
VEGFR-2 was found to be critical for VEGF-induced actin reorganization and
migration but did not affect mitogenicity in endothelial and tumour cells. We were
interested to gain insights on the binding mode by means of high and low-resolution
structural biology methods. Initial size exclusion chromatography (SEC) and MALS
analysis verified TSAd-VEGFR-2 interaction in vitro. SAXS analysis of the isolated
binding partners and the complex revealed that pY951 mediated binding of TSAd
resulted in an elongated multiprotein complex in which the binding partners orient in
a presumably parallel orientation. ---------- Zusammenfassung: Die Familie der vaskularen endothelialen Wachstumsfaktoren (VEGFs) spielt eine
entscheidende Rolle bei der Entwicklung und Aufrechterhaltung des Blut- und
Lymphgefasssystems. Die Familie besteht aus VEGF-A, -B, -C, -D und PLGF. Die
Wachstumshormone binden an drei Typ V Rezeptor-Tyrosin-Kinasen: VEGFR-1
(Flt1), VEGFR-2 (Flk1) und VEGFR-3 (Flt4). VEGFR-2 ist mehrheitlich fur die VEGF
induzierte Aktivierung von angiogenen und vaskulogenen Signalwegen und den
daraus resultierenden biologischen Effekten verantwortlich. Die VEGF-Rezeptoren
besitzen eine extrazellulare Domane bestehend aus sieben immunoglobulinahnlichen
Proteindomanen, die nebst der Rekrutierung von Liganden auch der
Dimerisierung des Rezeptors durch Ausbildung von homotypischen Kontakten dient.
Eine einzelne Transmembranhelix verbindet die extrazellulare Domane mit der
intrazellular geteilten Kinasedomane. Die Ligandenbindung an den Rezeptor fuhrt zu
Dimerisierung und der darauf folgenden Aktivierung durch Autophosphorylierung an
spezifischen Tyrosinen in der Kinasedomane. Die Phosphotyrosine rekrutieren
daraufhin Signalmolekule, die in der Lage sind spezifische Signalwege zu aktivieren.
Wir konnten zeigen, dass Glutaminsaure-Mutationen in der Transmembrandomane
von VEGFR-2 zu Konformationsanderungen in der Helix fuhren. Die daraus folgende
Neuausrichtung der Kinasedomanen resultierte in aktiven und inaktiven
Rezeptorkonformationen. Es gelang uns auch aufzuzeigen, dass die extrazellulare
Domane von VEGFR-2 eine wichtige Rolle bei der Blockierung des Rezeptors im
inaktiven Zustand in Abwesenheit des Liganden spielt. Hochaufgeloste
Strukturanalysen von isolierten Wildtyp und mutierten Transmembrandomanen
ergaben Strukturen, die hochstwahrscheinlich bei der Aktivierung des Rezeptors eine
essentielle Rolle spielen.
Ein zweites Projekt hatte zum Ziel, die Rolle der Kinase-Insertions-Domane (KID)
und der C-terminalen Domane (CD) bei der VEGFR-2 Aktivierung zu entschlusseln.
Wir konnten zeigen, dass die KID und die CD die VEGFR-2 Aktivierung regulieren.
Die KID und ein spezifisches Tyrosin in Position 951 innerhalb der KID waren
essentiell fur die Kinaseaktivierung. Deletion der CD fuhrte zu konstitutiver
Aktivierung von VEGFR-2. Wir denken, dass die CD den Rezeptor in Abwesenheit
des Liganden im inaktiven Zustand behalt. Mittels Small Angle X-ray Scattering
(SAXS) und Multi Angle Light Scattering (MALS) erhaltene Strukturdaten beweisen,
dass die Aktivierung der Kinasedomane von VEGFR-2 mit einer signifikanten
Anderungen in der Konformation einhergeht. Die aktive Kinasedomane nimmt eine
langliche, offene Konformation ein, wohingegen die Inaktive in einer globularen,
geschlossenen Konformation verbleibt. Sehr wahrscheinlich blockiert die CD dabei
das katalytische Zentrum des Enzyms, vergleichbar mit dem Mechanismus der von
der Tie-2 Kinasedomane verwendet wird. Sedimentation Equillibrium Analytical
Ultracentrifugation (AUC) Experimente haben gezeigt, dass die KID und die CD
wichtig sind, um die intrinsische Dimerisierungskapazitat der Kinasedomane zu
erhalten. Diese intrinsische Tendenz unterstutzt vermutlich die ligandeninduzierte
Dimerisierung bei der Rezeptoraktivierung. Deletionsmutanten zeigen eine niedrigere
Tendenz zur Dimerisierung mit steigender Proteinkonzentration. Experimente, die
zum Ziel hatten die Phosphorylierungskinetik der Kinasedomane zu untersuchen,
ergaben eine zeitlich geordnete sequentielle Aktivierungssequenz.
In einem dritten Projekt untersuchten wir die in vitro Interaktion von T-Zell
spezifischem Adapterprotein (TSAd) mit der aktiven Kinasedomane von VEGFR-2.
Es wurde gezeigt, dass die Y951 vermittelte Komplexbildung von TSAd mit VEGFR-2
zu VEGF induzierter Aktinreorganisation in den Zellen fuhrt. Die Endothel- und
Tumorzellen wurden dadurch zur Migration gebracht, wobei die Mitogenitat
unverandert blieb. Unser Ziel war es, mittels hoch- und niedrigauflosenden
strukturbiologischen Methoden spezifische Informationen zur Interaktion von TSAd
und VEGFR-2 abzuleiten. Erste chromatographische Analysen und Experimente
mittels Multi Angle Light Scattering (MALS) bewiesen die Interaktion der beiden
Proteine in vitro. SAXS Analysen der isolierten Bindungspartner und des Komplexes
ergaben einen langlichen Signalkomplex, in dem sich die Bindungspartner parallel
ausrichten.
The Omicron era of the COVID-19 pandemic commenced at the beginning of 2022 and whilst it started with primarily BA.1, it was latter dominated by BA.2 and related sub-lineages. Over the course of 2022, we monitored the potency and breadth of antibody neutralization responses to many emerging variants at two levels: (i) we tracked over 400,000 U.S. plasma donors over time through various vaccine booster roll outs and Omicron waves using sequentially collected IgG pools; (ii) we mapped the antibody response in individuals using blood from strigently curated vaccine and convalescent cohorts. In pooled IgG samples, we observed the maturation of neutralization breadth to Omicron variants over time through continuing vaccine and infection waves. Importantly, in many cases we observed increased antibody breadth to variants that were yet to be in circulation. Determination of viral neutralization at the cohort level supported equivalent coverage across prior and emerging variants with emerging isolates BQ.1.1, XBB.1 and BR.2.1 the most evasive. Further, these emerging variants were resistant to Evusheld, whilst neutralization resistance to Sotrovimab was restricted to BQ.1.1 and this was further supported by lack of antibody binding to this variant. An outgrowth advantage which correlated with better utilization of TMPRSS2 was observed across BQ lineages but not those derived from BA.2.75. We conclude at this current point in time that variants derived from BQ lineages can evade antibodies at levels equivalent to their most evasive BA.2.75 counterparts but sustain an entry phenotype that would promote an additional outgrowth advantage.Funding Information: This work was primarily supported by Australian Medical Foundation research grants MRF2005760 (ST, GM & WDR), Medical Research Future Fund Antiviral Development Call grant (WDR), the New South Wales Health COVID-19 Research Grants Round 2 (SGT) and the NSW Vaccine Infection and Immunology Collaborative (VIIM).Declaration of Interests: The authors declare no conflict of interest.Ethics Approval Statement: All human serum samples were obtained with written informed consent from the participants (2020/ETH00964; 2020/ETH02068; 2019/ETH03336; 2021/ETH00180; 2021/ETH0042). All primary isolates used herein were obtained from de-identified remnant diagnostic swabs that had completed all diagnostic testing under approval by the New South Wales Chief Health Officer following independent scientific review and as outlined in the ADAPT ethics protocol 2020/ETH00964.
Background: Continued phenotyping and ongoing surveillance are important in current and future monitoring of emerging SARS-CoV-2 lineages. Herein we developed pragmatic strategies to track the emergence, spread and phenotype of SARS-CoV-2 variants in Australia in an era of decreasing diagnostic PCR testing and focused cohort-based studies. This was aligned to longitudinal studies that span 4 years of the COVID-19 pandemic. Methods: Throughout 2023, we partnered with diagnostic pathology providers and pathogen genomics teams to identify relevant emerging or circulating variants in the New South Wales (NSW) community. We monitored emerging variants through viral culture, growth algorithms, neutralization responses and change entry requirements defined by ACE2 and TMPRSS2 receptor use. To frame this in the context of the pandemic stage, we continued to longitudinally track neutralisation responses at the population level using using sequential batches of pooled Intravenous Immunoglobulins (IVIG) derived from in excess of 700,000 donations. Findings: In antibodies derived from recent individual donations and thousands of donations pooled in IVIGs, we observed continued neutralization across prior and emerging variants with EG.5.1, HV.1, XCT and JN.1 ranked as the most evasive SARS-CoV-2 variants. Changes in the type I antibody site at Spike positions 452, 455 and 456 were associated with lowered neutralization responses in XBB lineages. In longitudinal tracking of population immunity spanning three years, we observed continued maturation of neutralization breadth to all SARS-CoV-2 variants over time. Whilst neutralization responses initially displayed high levels of imprinting towards Ancestral and early pre-Omicron lineages, this was slowly countered by increased cross reactive breadth to all variants. We predicted JN.1 to have a significant transmission advantage in late 2023 and this eventuated globally at the start of 2024. We could not attributed this advantage to neutralization resistance but rather propose that this growth advantage arises from the preferential utilization of TMPRSS2 cleavage-resistant ACE2.Interpretation: The emergence of many SARS-CoV-2 lineages documented at the end of 2023 to be initially associated with lowered neutralization responses. This continued to be countered by the gradual maturation of cross reactive neutralization responses over time. The later appearance and dominance of the divergent JN.1 lineage cannot be attributed to a lack of neutralization responses alone, and we support its dominance to be the culmination of both lowered neutralization and changes in ACE2/TMPRSS2 entry preferences.Funding: This work was primarily supported by Australian Medical Foundation research grants MRF2005760 (SGT, GM & WDR), Medical Research Future Fund Antiviral Development Call grant (WDR), the New South Wales Health COVID-19 Research Grants Round 2 (SGT & FB) and the NSW Vaccine Infection and Immunology Collaborative (VIIM) (ALC). Variant modeling was supported by funding from SciLifeLab's Pandemic Laboratory Preparedness program to B.M. (VC-2022-0028) and by the European Union's Horizon 2020 research and innovation programme under grant agreement no. 101003653 (CoroNAb) to B.M. Declaration of Interest: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.Ethical Approval: Human research ethics approval for this study was granted by Lifeblood Research Ethics Committee (30042020) and for the ADAPT cohort St Vincent's Hospital (2020/ETH00964) with donor consent forms including a statement that blood donations may be used for research purposes. NSW Health facilitated provision of de-identified residual COVID-19 diagnostic swabs for use in this study to support public health response under the governance of Health Protection NSW.
Abstract The Omicron era of the COVID-19 pandemic commenced at the beginning of 2022 and whilst it started with primarily BA.1, it was latter dominated by BA.2 and related sub-lineages. Over the course of 2022, we monitored the potency and breadth of antibody neutralization responses to many emerging variants at two levels: (i) we tracked over 420,000 U.S. plasma donors over time through various vaccine booster roll outs and Omicron waves using sequentially collected IgG pools; (ii) we mapped the antibody response in individuals using blood from strigently curated vaccine and convalescent cohorts. In pooled IgG samples, we observed the maturation of neutralization breadth to Omicron variants over time through continuing vaccine and infection waves. Importantly, in many cases we observed increased antibody breadth to variants that were yet to be in circulation. Determination of viral neutralization at the cohort level supported equivalent coverage across prior and emerging variants with emerging isolates BQ.1.1, XBB.1, BR.2.1 and XBF the most evasive. Further, these emerging variants were resistant to Evusheld, whilst neutralization resistance to Sotrovimab was restricted to BQ.1.1 and XBF. We conclude at this current point in time that dominant variants can evade antibodies at levels equivalent to their most evasive lineage counterparts but sustain an entry phenotype that continues to promote an additional outgrowth advantage. In Australia, BR2.1 and XBF share this phenotype and are dominating across NSW and Victoria. Research in context Evidence before this study Up until the BA.5 wave in mid 2022, many global waves were seeded by dominant variants such as Delta, Omicron BA.1 and Omicron BA.2. Following resolution of the BA.5, was the emergence of a pool of BA.4/5 and BA.2.75 sub-lineages accumulating clusters of similar polymorphisms located with the Receptor Binding Domain (RBD) of the Spike glycoprotein. Although iterative changes in the Spike increased the ability of each variant to navigate existing neutralising antibodies, it was unclear if this alone was sufficient to provide an outgrowth advantage to any one variant to fuel major case waves in global communities with high vaccine uptake and/or infection. Added value of this study Prior studies on incoming variants in Australian quarantine, highlighted the potential for Australia to represent a unique mix of cocirculating variants. Following the resolution of the BA.5 Omicron wave, many globally circulating variants appeared early on and ranged from BA.2.75 lineages, recombinants XBB.1, and XBC.1 in addition to many BA.5 derived BQ.1 lineages. Two additional lineages, the recombinant XBF and the BA.2.75 derived BR.2.1 also appeared and were uniquely enriched in Australia. Using 14 primary clinical isolates covering a continuum of circulating variants in Australia, we resolved neutralisation responses of 110 donors stringently documented for their vaccine and infection status over time. In addition, we also tested the well clinical utilised clinical monoclonals Evusheld and Sotrovimab. In addition to tracking donors, we also tracked immunity at the population level, using pooled IgG samples over time. The latter samples were the sum of 420,000 US plasma donors covering time periods of high-booster uptake alongside and in addition to large case waves. Whilst the above resolved the impact of Spike changes in neutralisations, we also tested each variant with respect to the efficiency of TMPRSS2 use, as this significantly influences viral tropism across the respiratory tract. Implications of all the available evidence All variants analysed herein have undertaken a convergent trajectory in accumulating a similar cluster of Spike polymorphisms. Many variants, including BQ.1.1, XBB.1, XBF and BR.2.1 have accumulated key changes that now render neutralisation responses lower in all cohorts and are neutralisation resistant to Evusheld. Whilst sotrovimab retained neutralisation capacity of many variants, there was significant reduction for variants BQ.1.1 and XBF. Impact of Spike changes on TMPRSS2 use were mixed and only one variant, BQ.1.2, had equal to increased usage relative to its parent BA.5. Analysis of neutralisation at the population level over time revealed two key observations. Firstly, whilst variants converged and lowered neutralisation responses, this reduction was negated over time with increasing neutralisation breadth. Secondly, responses to a variant proceeded its appearance and global circulation. In conclusion, whilst many variants are appearing and iterative changes in the spike will challenge antibody responses, increasing breadth in the community over time has enabled sufficient coverage to presently emerging variants. Furthermore, with the exception of BQ.1.2, viral use of TMPRSS2 has not increased and as such viral tropism towards epithelial cells of the upper respiratory tract we predict will be maintained.
Vascular endothelial growth factors (VEGFs) regulate blood and lymphatic vessel development and homeostasis. VEGF receptor 2 (VEGFR-2) is the major receptor involved in vasculogenesis and angiogenesis and regulates endothelial cell survival, migration, and mitogenesis. Ligand-mediated receptor dimerization instigates transmembrane signaling, thereby promoting activation of the intracellular kinase domain. The intracellular part of the receptor comprises the juxtamembrane domain, the catalytic kinase domain, the kinase insert domain (KID), and the carboxy terminal domain (CD). Here we show that the CD inhibits VEGFR-2 activity in the absence of ligand, whereas the KID, particularly a tyrosine residue in this domain (Y951), is indispensable for downstream signaling by the activated kinase. Because of the lack of crystallographic data for the complete kinase domain, we applied size-exclusion chromatography, multiangle laser scattering, analytical ultracentrifugation, and small-angle X-ray scattering to build and functionally validate structural models. Our data show substantial conformational changes of the kinase when it is switched from the inactive, unphosphorylated state to the active, phosphorylated state. Finally, we structurally characterized recombinantly produced protein complexes between VEGFR-2 and T cell-specific adapter protein, a molecule involved in downstream signaling by VEGFR-2.
Genetically distinct viral variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been recorded since January 2020. The introduction of global vaccine programs has contributed to lower COVID-19 hospitalisation and mortality rates, particularly in developed countries. In late 2021, Omicron BA.1 emerged, with substantially altered genetic differences and clinical effects from other variants of concern. Shortly after dominating global spread in early 2022, BA.1 was supplanted by the genetically distinct Omicron lineage BA.2. A sub-lineage of BA.2, designated BA.5, presently has an outgrowth advantage over BA.2 and other BA.2 sub-lineages. Here we study the neutralisation of Omicron BA.1, BA.2 and BA.5 and pre-Omicron variants using a range of vaccine and convalescent sera and therapeutic monoclonal antibodies using a live virus neutralisation assay. Using primary nasopharyngeal swabs, we also tested the relative fitness of BA.5 compared to pre-Omicron and Omicron viral lineages in their ability to use the ACE2-TMPRSS2 pathway.Using low passage clinical isolates of Clade A.2.2, Beta, Delta, BA.1, BA.2 and BA.5, we determined humoral neutralisation in vitro in vaccinated and convalescent cohorts, using concentrated human IgG pooled from thousands of plasma donors, and licensed monoclonal antibody therapies. We then determined infectivity to particle ratios in primary nasopharyngeal samples and expanded low passage isolates in a genetically engineered ACE2/TMPRSS2 cell line in the presence and absence of the TMPRSS2 inhibitor Nafamostat.Peak responses to 3 doses of BNT162b2 vaccine were associated with a 9-fold reduction in neutralisation for Omicron lineages BA.1, BA.2 and BA.5. Concentrated pooled human IgG from convalescent and vaccinated donors and BNT162b2 vaccination with BA.1 breakthrough infections were associated with greater breadth of neutralisation, although the potency was still reduced 7-fold across all Omicron lineages. Testing of clinical grade antibodies revealed a 14.3-fold reduction using Evusheld and 16.8-fold reduction using Sotrovimab for the BA.5. Whilst the infectivity of BA.1 and BA.2 was attenuated in ACE2/TMPRSS2 entry, BA.5 was observed to be equivalent to that of an early 2020 circulating clade and had greater sensitivity to the TMPRSS2 inhibitor Nafamostat.Observations support all Omicron variants to significantly escape neutralising antibodies across a range of vaccination and/or convalescent responses. Potency of therapeutic monoclonal antibodies is also reduced and differs across Omicron lineages. The key difference of BA.5 from other Omicron sub-variants is the reversion in tropism back to using the well-known ACE2-TMPRSS2 pathway, utilised efficiently by pre-Omicron lineages. Monitoring if these changes influence transmission and/or disease severity will be key for ongoing tracking and management of Omicron waves globally.This work was primarily supported by Australian Medical Foundation research grants MRF2005760 (ST, GM & WDR), MRF2001684 (ADK and ST) and Medical Research Future Fund Antiviral Development Call grant (WDR), Medical Research Future Fund COVID-19 grant (MRFF2001684, ADK & SGT) and the New South Wales Health COVID-19 Research Grants Round 2 (SGT).
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