Biology subjects

Eggink, D.

Publications and source records attributed to Eggink, D..

5 recordsLinked to original sources

bioRxiv2022

Bispecific antibodies combine breadth, potency, and avidity of parental antibodies to neutralize sarbecoviruses

SARS-CoV-2 mutational variants evade humoral immune responses elicited by vaccines and current monoclonal antibody (mAb) therapies. Novel antibody-based treatments will thus need to exhibit broad neutralization against different variants. Bispecific antibodies (bsAbs) combine the specificities of two distinct antibodies into one antibody taking advantage of the avidity, synergy and cooperativity provided by targeting two different epitopes. Here we used controlled Fab-arm exchange (cFAE), a versatile and straightforward method, to produce bsAbs that neutralize SARS-CoV and SARS-CoV-2 variants, including Omicron and its subvariants, by combining potent SARS-CoV-2-specific neutralizing antibodies with broader but less potent antibodies that also neutralize SARS-CoV. We demonstrate that the parental IgGs rely on avidity for their neutralizing activity by comparing their potency to bsAbs containing one irrelevant "dead" Fab arm. We used single particle mass photometry to measure formation of antibody:spike complexes, and determined that bsAbs increase binding stoichiometry compared to corresponding cocktails, without a loss of binding affinity. The heterogeneous binding pattern of bsAbs to spike (S), observed by negative-stain electron microscopy and mass photometry provided evidence for both intra- and inter-spike crosslinking. This study highlights the utility of cross-neutralizing antibodies for designing bivalent or multivalent agents to provide a robust activity against circulating variants, as well as future SARS-like coronaviruses.

immunology↗

bioRxiv2022

Broad SARS-CoV-2 Neutralization by Monoclonal and Bispecific Antibodies Derived from a Gamma-infected Individual

The worldwide pandemic caused by SARS-CoV-2 has remained a human medical threat due to the continued evolution of multiple variants that acquire resistance to vaccines and prior infection. Therefore, it is imperative to discover monoclonal antibodies (mAbs) that neutralize a broad range of SARS-CoV-2 variants for therapeutic and prophylactic use. A stabilized autologous SARS-CoV-2 spike glycoprotein was used to enrich antigen-specific B cells from an individual with a primary Gamma variant infection. Five mAbs selected from those B cells showed considerable neutralizing potency against multiple variants of concern, with COVA309-35 being the most potent against the autologous virus, as well as against Omicron BA.1 and BA.2. When combining the COVA309 mAbs as cocktails or bispecific antibody formats, the breadth and potency was significantly improved against all tested variants. In addition, the mechanism of cross-neutralization of the COVA309 mAbs was elucidated by structural analysis. Altogether these data indicate that a Gamma-infected individual can develop broadly neutralizing antibodies.

immunology↗

bioRxiv2022

IgG1 responses following SARS-CoV-2 infection are polyclonal and highly personalized, whereby each donor and each clone displays a distinct pattern of cross-reactivity against SARS-CoV-2 variants

Using a recently introduced efficient mass spectrometry-based approach we monitored individual donors IgG1 clonal responses in molecular detail, examining SARS-CoV-2 spike-protein-specific IgG1 repertoires. We monitored the plasma clonal IgG1 profiles of 8 donors (4 male and 4 female) who had recently experienced an infection by either the wild type Wuhan Hu-1 virus or one of 3 VOCs (Alpha, Beta and Gamma). In these donors we charted the full plasma IgG1 repertoires as well as the IgG1 repertoires targeting the SARS-CoV-2 spike protein trimer as antigen. We observed that shortly after infection in between <0.1% to almost 10% of all IgG1 antibody molecules present in plasma did bind to the spike protein. Each donor displayed a unique plasma IgG1 repertoire, but also each donor displayed a unique and polyclonal antibody response against the SARS-CoV-2 spike-protein variants. Our analyses revealed that certain clones exhibit (alike) binding affinity towards all four tested spike-protein variants, whereas other clones displayed strong unique mutant-specific affinity. We conclude that each infected person generates a unique polyclonal response following infection, whereby some of these clones can bind multiple viral variants, whereas other clones do not display such cross-reactivity. In general, by assessing IgG1 repertoires following infection it becomes possible to identify and select fully matured human plasma antibodies that target specific antigens, and display either high specificity or cross-reactivity versus mutated versions of the antigen, which will aid in selecting antibodies that may be developed into biotherapeutics.

immunology↗

bioRxiv2021

A SARS-CoV-2 mini-genome assay based on negative-sense RNA to study replication inhibitors and emerging mutations

Severe Acute Respiratory Coronavirus 2 (SARS-CoV-2) is a positive-sense single-stranded RNA virus and the causative agent of the Coronavirus disease 2019 (COVID-19) pandemic. Efforts to identify inhibitors of SARS-CoV-2 replication enzymes and better understand the mechanisms underlying viral RNA synthesis have largely relied on biosafety level 3 (BSL3) laboratories, limiting throughput and accessibility. Recently, replicon systems have been proposed that involve ~30 kb RNA-based replicons or large plasmids that express the viral structural and non-structural proteins (nsp) in addition to a positive-sense reporter RNA. Unfortunately, these assays are not user-friendly due to plasmid instability or a poor signal to background ratio. We here present a simple mini-genome assay consisting of a ~2.5 kb-long negative-sense, nanoluciferase-encoding sub-genomic reporter RNA that is expressed from a plasmid, and amplified and transcribed by the SARS-CoV-2 RNA polymerase core proteins nsp7, nsp8 and nsp12. We show that expression of nsp7, 8 and 12 is sufficient to obtain robust positive- and negative-sense RNA synthesis in cell culture, that addition of other nsps modulates expression levels, and that replication of the reporter RNA can be inhibited by active site mutations in nsp12 or the SARS-CoV-2 replication inhibitor remdesivir. The mini-genome assay provides a signal that is 170-fold above background on average, providing excellent sensitivity for high-throughput screens, while the use of small plasmids facilitates site-directed mutagenesis for fundamental analyses of SARS-CoV-2 RNA synthesis. Importance statementThe impact of the COVID-19 pandemic has made it essential to better understand the basic biology of SARS-CoV-2, and to search for compounds that can block the activity of key SARS-CoV-2 replication enzymes. However, studies with live SARS-CoV-2 require biosafety level 3 facilities, while existing replicon systems depend on long positive-sense subgenomes that are often difficult to manipulate or produce a high background signal, limiting drug-screens and a rapid analysis of emerging SARS-CoV-2 mutations during the COVID-19 pandemic. To make it easier to study emerging SARS-CoV-2 mutants and screen for inhibitors, we developed a simple mini-replicon that produces a minimal background signal, that can be used in any tissue culture lab, and that only requires four small plasmids to work.

microbiology↗

bioRxiv2021

The within-host evolutionary dynamics of seasonal and pandemic human influenza A viruses in young children

The evolution of influenza viruses is fundamentally shaped by within-host processes. However, the within-host evolutionary dynamics of influenza viruses remain incompletely understood, in part because most studies have focused on within-host virus diversity of infections in otherwise healthy adults based on single timepoint data. Here, we analysed the within-host evolution of 82 longitudinally-sampled individuals, mostly young children, infected with A/H3N2 or A/H1N1pdm09 viruses between 2007 and 2009. For A/H1N1pdm09 infections during the 2009 pandemic, nonsynonymous changes were common early in infection but decreased or remained constant throughout infection. For A/H3N2 viruses, early infection was dominated by purifying selection. However, as infections progressed, nonsynonymous variants increased in frequencies even though within-host virus titres decreased, leading to the maintenance of virus diversity via mutation-selection balance. Our findings suggest that this maintenance of genetic diversity in these children combined with their longer duration of infection may provide important opportunities for within-host virus evolution.

microbiology↗