Southwestern France proved to be a happy hunting ground for British athletes on Saturday at the second edition of Challenge Vieux Bocau but four of the six podium spots were taken by the travelling British contingent Victory on Saturday represented a first professional middle-distance victory for Jack Hutchens It has come off the back of a year in which he has had dual objectives Hutchens swam close to the front pack of swimmers biked strongly to break away with a small group including fellow Brit “I thought Harry is going to run away with it but I just had the day of my life and I am so happy.” Daisy Davies has been mixing things up this year A recent winner of the draft-legal British Elite Championships at Mallory Park Davies also raced and won at Challenge Wales in June this year to show talents across multiple formats and distances Heading a British quarter which included Chloe Sparrow Rosie Weston and Megan McDonald out of the opening 1.9km swim and built herself a healthy lead of almost five minutes over McDonald at T2 with Sweden’s Anna Bergsten a further minute back While Bergsten (1:20:42) and McDonald (1:23:33) took time back – the Swede moving into second place in the process – but Davies had set up her race on the second discipline and had more than enough in hand to take a second Challenge Family win “It was a great event and the crowd were awesome A credit to the volunteers and organisers to put on such a great event,” Saturday 5 October 2024 – 1.9km / 90km / 21.1km photographer José Luis Hourcade (www.joseluishourcade.com) was on hand to capture the race in pictures Follow the madness of the 'Race that eats its young' on RUN247 24-year-old Draper finished fourth in a very strong pro field Professional triathlete Will Draper produced a fantastic result at the Challenge Vieux Boucau event in southwestern France over the weekend Renowned for its unique landscape, Vieux-Boucau-les-Bains is a wild west-coast family resort lined with sand dunes and popular with surfers. It also has a unique, sheltered marine lake without waves or current, perfect for the 1.9km saltwater swim. This was followed by a single lap 90km bike leg along fast, flat roads and through the forests of the Landes region. The run course was round the marine lake, finishing in the centre of Vieux Boucau. Delighted with his performance, Draper posted the following message on Instagram: ‘Worked hard all day for that one, one of my best final hour’s on the bike into one of my best half marathons off the bike. ‘Still managing the ankle injury but definitely felt like my old self on that run. The fire was burning!’ On Facebook he added: ‘It was a tight race among the top 10, with the British well in the mix. ‘The front of the race had a lot of motorbikes offering draft, which definitely effected my ride and meant I wasn’t able to catch guys who I have previously out rode by minutes! ‘I clocked a 25-minute swim (1min 18sec per 100 metres) into a 2hr 00m 12s bike (45kph) and a 1hr 11m 16s half marathon (3m 24s per km) for a total 70.3 mile time of 3h 40m 05s. ‘75 seconds faster in the swim will change my whole race. Probably going to do around 35-40km of swimming per week this winter and is likely to not happen until 2026 even with daily commitment.’ He ended by thanking everyone who supported or tracked him during the event in France. Competing in the Manchester Half-Marathon this Sunday (Alan Corlett is also going over for that event), Draper’s next triathlon is the Challenge Paguera in Mallorca a week on Saturday, October 19. Comments Tel: 01624 695695[email protected]Follow us Further Links Owned or licensed to Tindle Newspapers Ltd | Independent Family-Owned Newspapers | Copyright & Trade Mark Notice & 2013 - 2025 This is a preview of the inaugural race – click here to read about dramatic wins for Mathis Margirier and Caroline Pohle Challenge Vieux Boucau will mark the end of a Challenge Family triathlon triple header this weekend, and we have stellar pro fields including Alistair Brownlee Dorian Coninx and Imogen Simmonds among others We have already had terrific action provided by back-to-back middle distance races won by Youri Keulen and the aforementioned Simmonds at Challenge Barcelona and Challenge Mallorca Simmonds returns for her third race in as many weeks as does double Olympic Champion Brownlee – who picked up back-to-back podiums in Barcelona and Mallorca after his return from ankle surgery We have all the information you need on start times and how to track your favourite athletes in our preview of the men and women’s fields you can find out who to keep an eye out for on race day This weekend’s racing will take place on Saturday October 21 04:00 on the East Coast of the United States and 01:00 PST For live tracking updates to find out how your favourite athletes are doing on race day, you can click on this link.  Leading the way on home soil in Vieux Boucau is Frenchman Mathis Margirier who finished second last weekend in Mallorca He will be chasing his fifth Challenge Family podium of the year Fourth at the IRONMAN 70.3 World Championship in August Margirier will get the chance to line up against two of the men who kept him off the podium in Finland as German duo Frederic Funk and Rico Bogen start who didn’t finish in Mallorca last weekend will be aiming to complete his first race since becoming world champion who finished second in Barcelona and third in Mallorca faces stiff competition in France in the fight for another podium Bogen will not be the only world champion in the field as newly-crowned WTCS World Champion Coninx races over the middle distance for the first time this season With the swim speed to make the front pack it will be interesting to see how the Olympian holds up Having won those back-to-back races in Barcelona and Mallorca Swiss star Simmonds will be the heavy favourite to take a third successive win in France this weekend as the PTO World #10 competes for her seventh podium of the season Having won by almost three minutes in both Spanish events Simmonds has yet to be pushed to the line this month and despite some strong competition in Vieux Boucau is the heavy favourite to be the runaway victor yet again on Saturday Home favourite Emilie Morier took a big scalp at the start of the season when she beat Australian Ashleigh Gentle at the Peniscola Infititri but was way down in 30th place at the IRONMAN 70.3 World Championship in August is another podium contender but neither athlete should threaten Simmonds Lizzie Rayner is among a handful of names who could compete for the podium who beat Morier last month at IRONMAN 70.3 Knokke-Heist where she finished second Unfortunately, your version of Internet Explorer is outdated and it doesn't support some of the technologies used on this site. This may lead to incorrect display of some pages. Please upgrade your browser and get all benefits of browsing the new JINR site You can also use the previous version of the JINR site On 5 August 2018, at 6:00 PM, a concert will be delivered by Alain Blesing and Claudie Boucau (France) in the JINR Universal Public Library A family duo that combines the most unimaginable opposites theatrical music for children and root French prog-rock acoustic flute and guitar processor electronics structured multipart pieces of music for a large ensemble at stages of leading European festivals and “open” co-creation with unfamiliar musicians in Siberian villages Alain and Claudi’s duo provides a diverse programme that combines authors’ pieces (from the “Jog” Indian melody based pieces to chamber dissonance melodies) jazz standards of various ranges of recongition (starting from «Goodbye Pork-Pie Hat» and ending with complex pieces by Dave Holland of the late period) and spontaneous improvisation Blokhinka in Facebook Event in VK Metrics details Here we show that about 6% of blood monocytes of patients with COVID-19 are infected with SARS-CoV-2 Monocyte infection depends on the uptake of antibody-opsonized virus by Fcγ receptors The plasma of vaccine recipients does not promote antibody-dependent monocyte infection SARS-CoV-2 begins to replicate in monocytes and infectious virus is not detected in the supernatants of cultures of infected monocytes infected cells undergo pyroptosis mediated by activation of NLRP3 and AIM2 inflammasomes but not infected epithelial and endothelial cells from lung autopsies from patients with COVID-19 have activated inflammasomes these findings suggest that antibody-mediated SARS-CoV-2 uptake by monocytes and macrophages triggers inflammatory cell death that aborts the production of infectious virus but causes systemic inflammation that contributes to COVID-19 pathogenesis The plots in g show the median (centre line) the interquartile range between the 25th and 75th percentiles (box) and the 25th percentile value − 1.5× the interquartile range (lower whisker) and  the 75th percentile value + 1.5× the interquartile range (upper whisker) Statistical analysis was performed using two-tailed nonparametric unpaired t-tests (b one-way analysis of variance (ANOVA) with Tukey multiple-comparisons test (e two-tailed nonparametric unpaired t-tests (g) and two-way ANOVA with Tukey multiple-comparisons test (h); *P < 0.05 IL-1RA and IL-18 were all elevated in the samples from patients with severe disease compared with those with mild or moderate disease but the increase in GSDMD was not significant these results suggest ongoing pyroptosis in COVID-19 blood that was more prominent in severe disease non-COVID-19 patients or patients with COVID-19 at the time of presentation were analysed by imaging flow cytometry for ASC caspase-1 activation (FLICA) and/or Zombie dye uptake The percentage of monocytes with activated ASC (a) or caspase-1 (b) (n = 8 (HD) n = 10 (COVID-19)) or colocalized ASC/caspase-1 specks (c) (n = 8 (HD) Representative images (top) and quantification of all samples (bottom) are shown The percentage of ASC-speck-containing monocytes with colocalized activated caspase-1 Representative images of ASC (e) or Zombie dye (f) and GSDMD co-stained monocytes Lysates of purified monocytes of HDs and patients with COVID-19 and of LPS- and nigericin-treated monocytes of HDs (+) probed with a monoclonal antibody that recognizes full length GSDMD (GSDMD-FL) and the C-terminal of GSDMD (GSDMD-CT) (top) Representative of n = 4 independent experiments Representative images of ASC co-staining with NLRP3 (left; n = 5 (HD) n = 4 (COVID-19)) and pyrin (right; n = 4 (HD) and quantification of monocytes showing ASC specks colocalized with the indicated inflammasomes (i) Representative images of co-staining of ASC Statistical analysis was performed using one-way ANOVA with Tukey multiple-comparisons test (a–d) and two-way ANOVA with Tukey multiple-comparisons test (i); *P < 0.05 GSDMD-FL was detected in all of the HD samples but in only 1 out of 3 samples from patients with COVID-19 GSDMD-CT was detected in monocytes of patients with COVID-19 and the positive control (LPS + nigericin-treated HD monocytes) Although COX-IV was detected in all of the samples full-length β-actin was not detected in one COVID-19 sample but β-actin fragments were detected in all of the samples from patients with COVID-19 and in nigericin-activated HD monocytes monocytes of patients with COVID-19 are undergoing pyroptosis indicate that COVID-19 monocytes die of pyroptosis Digital scanner images of a representative patient who experienced trauma (left) and a patient with COVID-19 (middle) showing a magnified image of representative infected CD14+ (top) and CD14− (bottom) cells from the lungs of the patient with COVID-19 (right) Representative confocal microscopy COVID-19 lung images of infected CD14+ (j) and CD14− (k) cells (left) quantification of CD14+ (j) and CD14− (k) cells that are N positive and/or have ASC specks in the lungs of patients with COVID-19 (n = 5) and control individuals (n = 3) representative images of CD14−N+ cells (left) were co-stained for ASC and E-cadherin Statistical analysis was performed using two-tailed nonparametric unpaired t-tests (Mann–Whitney U-tests) (b–d f–h) and two-way ANOVA with Tukey multiple-comparisons test (j ASC specks were not seen in control autopsies About a quarter of CD14+ lung cells had ASC specks although only approximately 8% were N positive suggesting that danger-associated molecular patterns released from infected or otherwise damaged lung cells may have activated inflammasomes in uninfected macrophages HD monocytes (n = 3) were primed (black bars) or not (white bars) with LPS infected with icSARS-CoV-2-mNG and stained 48 h later for N and ASC Virus was preincubated with IgG1 control mAb114 non-neutralizing anti-spike (C1A-H12) or neutralizing anti-RBD (C1A-B12) or with pooled plasma from patients with COVID-19 and these were retained throughout culture Representative imaging flow cytometry images of uninfected (top) N+NG− (middle) or N+NG+ (bottom) monocytes Quantification of the percentage of ASC speck+ (b) or N+ (c) monocytes LPS-activated HD monocytes were infected with icSARS-CoV-2-mNG preincubated with pooled COVID-19 plasma depleted or not depleted of immunoglobulins using protein A/G beads (n = 3; e) or preincubated with pooled plasma from HDs non-COVID-19 patients or patients with COVID-19 with mild and/or severe disease (n = 3; f pooled from patients with COVID-19 of mixed severity (n = 3) or patients with COVID-19 with low (about 8%) or high (about 30%) afucosylated (Afucos.) anti-spike IgG (n = 11) (h LPS-treated HD monocytes were infected with icSARS-CoV-2-mNG preincubated with pooled plasma from patients with COVID-19 depleted or not depleted of IgG or IgA as indicated in the presence of the indicated blocking or isotype control (Iso) antibodies (n = 3; j k) or antiviral drugs (l (10 µM remdesivir) and infection was assessed 48 h later by NG fluorescence The statistical analysis in m compared drug with no drug RT–qPCR analysis of genomic SARS-CoV-2 N RNA (n) and sgRNA (o left) in uninfected or infected HD monocytes (n = 3) Infected HEK293T cells were used as a positive control (n = 3) Agarose gel electrophoresis of ethidium-bromide-stained RT–qPCR-amplified sgRNA is shown (o The approximately 1,600-bp band in the samples from patients with COVID-19 was sequenced and confirmed to be N sgRNA SARS-CoV-2 plaque-forming units (PFU) in culture supernatants of infected monocytes (Mono) or Vero E6 cells collected at the indicated hours post-infection (h.p.i.) Statistical analysis was performed using two-way ANOVA with Sidak multiple-comparisons test (b–d) two-tailed nonparametric unpaired t-tests (e) and one-way ANOVA with Tukey multiple-comparisons test (f–p); *P < 0.05 Data are representative of n = 3 replicate experiments pooled plasma from non-COVID-19 patients slightly increased infection suggesting possible inefficient viral uptake by some non-COVID plasma component Disease severity did not affect infection by the plasma of patients with COVID-19 as pooled mild and/or severe plasma similarly facilitated infection suggesting that an Ig-independent plasma component might facilitate infection Blocking CD16 or CD64 or IgG depletion strongly inhibited infection whereas blocking the other receptors had no significant effect The combination of anti-CD16 and anti-CD64 blocking antibodies did not inhibit virus uptake more than either blocking antibody on its own SARS-CoV-2 infection of monocytes is mostly mediated by CD16 and/or CD64 uptake of opsonized virus Infection was not detected in neutrophils of patients with COVID-19 The most abundant amplified sgRNA fragment migrated on agarose gels at the size of the N sgRNA (1,560 nucleotides) and its identity was confirmed by sequencing plaques were easily detected in culture supernatants from infected Vero cells collected at 48 h after infection monocyte infection did not produce infectious virus Pyroptotic myeloid cells are probably a major cause of the serious inflammatory sequelae that lead to acute lung injury vascular leak and respiratory distress in patients with severe disease patients with severe COVID-19 had increased plasma biomarkers of pyroptosis compared with patients with mild or moderate COVID-19 neither antibody titres nor the proportion of infected ASC-speck-positive monocytes at presentation correlated with severe disease perhaps because of the small number of samples Larger cohorts are needed to better assess the relative importance of monocyte/macrophage pyroptosis in severe COVID-19 pathogenesis The large numbers of infected monocytes and macrophages the fact that a quarter of lung macrophages have activated inflammasomes and that myeloid cells are the major source of IL-1 and other inflammatory cytokines make it probable that monocyte/macrophage infection and inflammasome activation are important in severe COVID-19 pathogenesis Although neutrophils could potentially be infected infection of freshly isolated COVID-19 neutrophils or in vitro-infected HD neutrophils was not detected neutrophil infection is probably not a major contributor to pathogenesis although neutrophil activation of GSDMD-dependent NETosis (a cell death process involving neutrophil extracellular traps (NETs)) or other features of neutrophil activation may well be important drivers It will be worthwhile to study other infected cells as potential sources of inflammation and to understand what aspects of monocyte/macrophage activation enhance infection including the dominant classical subtype that is not infected whereas CD16 is more selectively expressed and all the infected patient monocytes are CD16 positive This means that CD16 is probably the major Fc receptor that mediates viral entry into monocytes Blocking infection by anti-CD64 antibodies may be indirect as CD64 and CD16 use the same signalling adaptors and associate on the cell surface which has overlapping features with severe COVID-19 disease these drugs strongly improved survival and reduced plasma IL-6 and TNF IgG isolated from patients with COVID-19 with a higher proportion of afucosylated antibodies significantly increased in vitro monocyte infection but IgG from patients with fewer afucosylated antibodies did not The increased pathogenicity of afucosylated antibodies could be secondary to antibody-mediated infection and downstream inflammasome activation in monocytes and macrophages our findings about afucosylation are preliminary and more work is needed to make this association alter protective versus deleterious functions of anti-spike antibodies will be important not only for understanding SARS-CoV-2 pathogenesis but also for choosing the best preparations of convalescent patient plasma and monoclonal antibodies for therapy and/or prevention of severe disease HD samples were processed and analysed in parallel with the patient samples The participants were enrolled from 9 July 2020 to 10 January 2021 at Boston Children’s Hospital (BCH) with IRB-approved waiver of informed consent who received two doses of the Pfizer-BioNtech mRNA vaccine were enrolled 3 weeks after the second dose and their plasma was pooled to evaluate whether it promoted monocyte infection Lung samples from five individuals who died from COVID-19 (Supplementary Table 3) and three individuals who died from trauma and without lung disease were obtained from MGH The study was approved by the institutional review board of MGH IRB 2020P001147 Informed consent was obtained from the relatives of study participants Lung tissue specimens were obtained within 24 h of autopsy and immediately formalin-fixed and embedded in paraffin A list of reagents and antibodies and their sources is provided in Supplementary Table 4 Samples were processed using the recommended safety precautions in a BSL-2+ facility Blood tubes were centrifuged at 2,000 rpm for 10 min to separate the plasma from blood cells The plasma was collected in a new tube and incubated or not with 1% Triton X-100 for 1 h on ice before aliquoting and freezing at −80 °C Blood cells were resuspended in PBS and layered over Ficoll for density centrifugation PBMCs were collected from the interface and subjected to red blood cell lysis (if necessary) with Red Blood Cell Lysing Buffer Hybri-Max for 5 min on ice followed by quenching with RPMI medium supplemented with 10% FBS and 1% penicillin–streptomycin PBMCs were washed once more with RPMI and one fraction was stained for flow cytometry while the remaining cells were used for monocyte purification by negative selection using the RosetteSep Human Monocyte Enrichment Cocktail Neutrophils of patients with COVID-19 were isolated from the whole blood by immunomagnetic negative selection using the EasySep Direct Human Neutrophil Isolation Cocktail according to the manufacturer’s instructions HD monocytes for in vitro infection were purified from PBMCs by positive selection with CD14+ magnetic beads The red blood cell pellet from the Ficoll density centrifugation was used to isolate neutrophils from the same HD samples Neutrophils were separated from the RBC pellet by hypotonic lysis The THP-1 monocytic cell line and Vero E6 cells were obtained from ATCC A549 cells and HEK293T cells overexpressing ACE2 were obtained from the MassCPR variants repository at Ragon Institute ACE2 expression was validated by RT–qPCR and anti-ACE2 flow cytometry All cells were tested for mycoplasma contamination IFNβ and IFNγ were measured in plasma samples using a custom Luminex assay (R&D Systems) according to the manufacturer’s instructions Sample data were acquired using the Luminex xPONENT 4.2 for MAGPIX Analyzer at the Analytical Instrumentation Core Lab of Boston University and analysed with Milliplex Analyst v5 The plasma levels of IL-1β were measured using the Simple Plex cartridge Ella (ProteinSimple) according to the manufacturer’s instructions at the BCH All of the samples were diluted 1:3 with the dilution buffer and the analytical performance was conducted on the ProteinSimple Ella automated immunoassay platform (Bio-Techne) The samples were acquired using the Simple Plex Runner v.3.7.2.0 software and analysed using Simple Plex Explorer 3.7.2.0 GSDMD was measured in the same samples using the Human GSDMD ELISA kit (MyBiosource) according to the manufacturer’s instructions and LDH activity was measured using the CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega) Results from the latter assays were analysed using the Biotek Synergy 2 analyzer; GSDMD absorbance was measured at 450 nm and LDH absorbance was measured at 490 nm Absorbance levels were quantified by linear regression based on the standard curve The enzyme-linked immunosorbent assay (ELISA) anti-spike RBD kit (BioLegend) was used to quantify antigen-specific IgG in the plasma from HDs non-COVID-19 patients and patients with COVID-19 ELISA was performed according to the manufacturer’s instructions Anti-spike RBD absorbance was measured at 450 nm and 570 nm and quantified by linear regression based on the standard curve Fixed monocytes were permeabilized with 0.1% Triton X-100 for 10 min and washed twice with PBS + 3% FBS Monocytes were then blocked for 30 min with PBS + 5% FBS washed twice and then stained with unconjugated primary antibodies against ASC (1:200 mouse) (1:500) or SARS-CoV-2 nucleocapsid protein (1:500 followed by three washes with PBS + 3% FBS The cells were then stained with secondary antibodies (donkey anti-mouse rabbit or goat conjugated with Alexa Fluor 488 THP-1 cells treated with LPS + nigericin or transfected with Poly(dA:dT) using Lipofectamine 2000 and HEK293T cells (negative control) were stained with anti-NLRP3 and anti-AIM2 antibodies for antibody validation cells were fixed and then stained with DAPI (1:1,000) for 10 min washed three times and cytospun onto glass slides (VWR) and sealed using polyvinyl alcohol and 1.5 mm coverslips (VWR) Confocal images were acquired using the Zeiss LSM 800 system with 405-nm 561-nm and 633-nm lasers (emission filters respectively) and a ×40 or ×63 1.4 NA oil-immersion objective Images were acquired using Zen Black 2.0 and processed using Zen Blue 3.2 cells were resuspended in PBS + 3% FBS for analysis Data were acquired using the ImageStream X MKII system with ×60 magnification (Amnis) the INSPIRE v.2 acquisition software and were analysed using IDEAS v.6.2 (Amnis) Monocytes were gated based on area/aspect ratio AIM2 and pyrin specks were gated and quantified on the basis of fluorophore intensity/maximum pixels PBMCs were washed and stained for viability with Zombie Yellow in PBS (1:200) for 15 min on ice centrifuged and then stained with anti-annexin V PE (1:200) antibodies in 1× annexin buffer for 15 min on ice cells were blocked for 10 min with anti-CD32 (1:100) in PBS + 3% FBS and then stained for 15 min on ice with a cocktail of antibodies to identify lymphocyte and myeloid cell subsets (all 1:200 except CD19 BV650 Purified monocytes and an A549 cell line overexpressing ACE2 were blocked with anti-CD32 then stained with primary antibodies for ACE2 (1:100) for 15 min on ice The secondary anti-goat AF488 antibody was co-incubated with anti-CD14 PE-Cy7 (1:200) and anti-CD147 APC (1:100) antibodies cells were resuspended in 2% PFA and kept at 4 °C until flow cytometry analysis In vitro-infected monocytes were fixed and permeabilized with 0.1% Triton X-100 Cells were stained with primary antibodies for dsRNA (J2 then stained with secondary antibodies (donkey anti-mouse conjugated with Alexa Fluor 647 Cells were acquired using the FACS Canto II or LSR II using the FACSDiva v7 acquisition software and data were analysed using FlowJo v.10.7.1 Freshly isolated monocytes were washed and resuspended in RPMI 10% FBS with FLICA substrate (BioRad FAM-FLICA Caspase-1 kit) and cultured for 1 h at 37 °C Cells were then washed twice with 1× apoptosis buffer (from the kit) and fixed with 1× fixative (from the kit) Cells were kept at 4 °C until further staining and analysis Lysates of enriched monocytes from HDs and patients with COVID-19 the former treated or not for 16 h at 37 °C with 100 ng ml−1 LPS and 20 µM nigericin transferred to nitrocellulose membranes and blotted to detect GSDMD using (Abcam ab210070) primary rabbit monoclonal antibodies and secondary anti-rabbit IgG The membranes were also blotted for β-actin and COX-IV Formalin-fixed and paraffin-embedded lung parenchymal samples were stained for SARS-CoV-2 N and immunofluorescence was analysed on the Leica Bond RX automated staining platform using the Leica Biosystems Refine Detection Kit (Leica) The antibody for SARS nucleocapsid (Novus) was run with citrate antigen retrieval and tagged with Alexa Fluor 488 Tyramide (Life) the antibody for CD14 (Cell Signaling) was incubated and tagged with Alexa Fluor 594 Tyramide (Life) staining for ASC (Santa Cruz) was analysed using antibodies tagged with Alexa Fluor 647 Tyramide (Life) EDTA stripping was performed before anti-CD31 or anti-E-cadherin staining tagged to Alexa Fluor 555 Tyramide (Life) The slides were scanned using the Aperio Versa Digital Pathology Scanner (Leica) and analysed using Aperio ImageScope v.12.4.3 (Leica) The slides were also analysed by confocal microscopy as described above The NG fusion protein is expressed only during viral replication The SARS CoV-2 US-WA1/2020 ancestral (WA) variant was obtained from BEI Resources The B.1.617.1/Delta variant isolate was obtained from the MassCPR variant repository the variant was isolated at the Ragon BSL3 by rescue on Vero-E6 cells from primary clinical specimens The whole genome of subsequent viral stocks was sequenced to confirm that no additional mutation arose during virus expansion HD monocytes/neutrophils were purified from apheresis leukoreduction collars collected at Brigham and Women’s Hospital Monocytes were incubated overnight with medium or 100 ng ml−1 LPS SARS-CoV-2 (WA) and SARS CoV-2 B.1.617.1/Delta (multiplicity of infection (MOI) = 1) in a BSL-3 facility Infection of A549-ACE2 cells at an MOI of 0.01 was used as a control The viral inoculum was treated with 10 µg ml−1 of antibody (isotype control mAb114 or 5% pooled plasma (heat-inactivated or not; Ig-depleted or not patients with COVID-19 of mixed disease severity (n = 12 (total) n = 4 (severe)) or vaccinated HDs (n = 6) before infection with SARS-CoV-2 for 30 min at room temperature Treated virus (100 µl) was added to monocytes (2 × 106 cells per well) in 48-well plates Infected cells were incubated at 37 °C under 5% CO2 with gentle shaking every 10 min for 1 h after which the culture volume was increased to 500 µl with RPMI supplemented with 5% heat-inactivated normal AB human serum and 10 µg ml−1 of the aforementioned antibodies or 5% pooled plasma from HDs or patients with COVID-19 Cultures were then incubated at 37 °C under 5% CO2 for 48 h at which time the cells were collected and fixed for 20 min with 4% PFA and then stained monocytes were incubated at 37 °C under 5% CO2 for 1 h with 10 µM remdesivir (GS-5734) or camostat mesylate before infection To find an appropriate remdesivir concentration serial dilutions between 10 and 80 µM were analysed To compare plasma obtained from patients with different disease severity plasma was pooled on the basis of the MGH acuity score (A1–A5) contained 8.4 ± 0.7% afucosylated IgG and high afucosylated samples IgG was also purified from pooled plasma from HDs and patients with COVID-19 using the Melon gel IgG Spin Purification Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions Virus was preincubated with 10 µg ml−1 of purified IgG and the infection was performed as described above sgRNA qPCR products were also analysed by electrophoresis on 1% agarose gels stained with ethidium bromide and visualized on the Chemidoc imager (BioRad) The approximately 1,600 nucleotide band was excised and sequenced to confirm its origin as the SARS-CoV-2 sgRNA encoding N Vero E6 cells were seeded as monolayers in 24-well plates 1 day before infection Virus-infected sample culture supernatants were serially diluted in DMEM The plates were washed once with DPBS and then infected with 100 µl of diluted sample and incubated at 37 °C under 5% CO2 for 1 h with rocking every 15 min the inoculum was removed and an overlay of 1% methylcellulose (Sigma-Aldrich) in complete MEM (Gibco) was applied to each well The plates were incubated at 37 °C until plaques were observable in positive control wells and the cell monolayer was fixed with 4% PFA and stained with crystal violet Plaques were then counted to quantify the virus titre in PFU per ml Statistical analysis was performed using GraphPad Prism v.9.0 Normal distribution of the data was evaluated using the D’Agostino and Pearson normality test before applying statistical methods Distributions were considered to be normal if P ≤ 0.05 Parametric or nonparametric (Mann–Whitney U-test) two-tailed unpaired t-tests were used to compare two unpaired groups Multiple-group comparisons were analysed using one-way ANOVA with Sidak or Tukey multiple-comparisons tests or nonparametric Kruskal–Wallis with Dunn post-test Multiple groups were compared using two-way ANOVA with additional Sidak or Tukey multiple-comparisons test Mean plasma values from hospitalized patients with COVID-19 on each day were compared between severity groups by multiple unpaired t-tests Correlations of plasma levels were determined by simple linear regression and Pearson correlation coefficient Further information on research design is available in the Nature Research Reporting Summary linked to this paper The data and materials supporting the findings of this study are available from the corresponding authors on request Characteristics of SARS-CoV-2 and COVID-19 An inflammatory cytokine signature predicts COVID-19 severity and survival Channelling inflammation: gasdermins in physiology and disease Interleukin-1 in the pathogenesis and treatment of inflammatory diseases Risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease 2019 pneumonia in Wuhan Inflammasome activation at the crux of severe COVID-19 Antibody responses to SARS-CoV-2 in patients with COVID-19 Impaired type I interferon activity and inflammatory responses in severe COVID-19 patients Inflammasomes are activated in response to SARS-CoV-2 infection and are associated with COVID-19 severity in patients Inflammasome signaling and regulation of interleukin-1 family cytokines Longitudinal proteomic analysis of severe COVID-19 reveals survival-associated signatures SARS-CoV-2 N protein promotes NLRP3 inflammasome activation to induce hyperinflammation The cell biology of inflammasomes: mechanisms of inflammasome activation and regulation A tug-of-war between severe acute respiratory syndrome coronavirus 2 and host antiviral defence: lessons from other pathogenic viruses Calpain drives pyroptotic vimentin cleavage and cell rupture that mediates immunostimulation Gasdermin pores permeabilize mitochondria to augment caspase-3 activation during apoptosis and inflammasome activation A noncanonical function of cGAMP in inflammasome priming and activation Severe acute respiratory syndrome coronavirus 2-induced immune activation and death of monocyte-derived human macrophages and dendritic cells Song, X. et al. Little to no expression of angiotensin-converting enzyme-2 on most human peripheral blood immune cells but highly expressed on tissue macrophages. Cytometry A, https://doi.org/10.1002/cyto.a.24285 (2020) Human basigin (CD147) does not directly interact with SARS-CoV-2 spike glycoprotein No evidence for basigin/CD147 as a direct SARS-CoV-2 spike binding receptor CD147-spike protein is a novel route for SARS-CoV-2 infection to host cells five-marker alternative to CD16–CD14 gating to identify the three human monocyte subsets The role of IgG Fc receptors in antibody-dependent enhancement Dynamic changes in anti-SARS-CoV-2 antibodies during SARS-CoV-2 infection and recovery from COVID-19 Double-stranded RNA is produced by positive-strand RNA viruses and DNA viruses but not in detectable amounts by negative-strand RNA viruses Clark, S. A. et al. Molecular basis for a germline-biased neutralizing antibody response to SARS-CoV-2. Preprint at bioRxiv https://doi.org/10.1101/2020.11.13.381533 (2020) Proinflammatory IgG Fc structures in patients with severe COVID-19 Afucosylated IgG characterizes enveloped viral responses and correlates with COVID-19 severity High titers and low fucosylation of early human anti-SARS-CoV-2 IgG promote inflammation by alveolar macrophages Patients with COVID-19: in the dark-NETs of neutrophils SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor and innate immune responses of the coronavirus SARS-CoV-2 in human respiratory tract and conjunctiva: an analysis in ex-vivo and in-vitro cultures COVID-19-neutralizing antibodies predict disease severity and survival Phase separation drives RNA virus-induced activation of the NLRP6 inflammasome Human NLRP1 is a sensor for double-stranded RNA FDA-approved disulfiram inhibits pyroptosis by blocking gasdermin D pore formation Succination inactivates gasdermin D and blocks pyroptosis The potential danger of suboptimal antibody responses in COVID-19 Early treatment for COVID-19 with SARS-CoV-2 neutralizing antibody sotrovimab Early convalescent plasma for high-risk outpatients with COVID-19 Enhancement versus neutralization by SARS-CoV-2 antibodies from a convalescent donor associates with distinct epitopes on the RBD afucosylated antibody responses are associated with COVID-19 severity Division of Viral Diseases, NCIRD. 2019-Novel Coronavirus (2019-nCoV) Real-Time rRT-PCR Panel Primer and Probes, https://www.cdc.gov/coronavirus/2019-ncov/lab/rt-pcr-panel-primer-probes.html (CDC The architecture of SARS-CoV-2 transcriptome SARS-CoV-2 virus culture and subgenomic RNA for respiratory specimens from patients with mild coronavirus disease Download references We thank members of the MGH COVID-19 collection and the processing team (K Wang for providing the high and low afucosylated IgGs from donors with COVID-19; the members of the Analytical Instrumentation Core Lab of Boston University for running and analysing the Luminex Multiplex assay; D This research was supported by the Lemann Brazil Research Fund (to J.L and C.J.); the National Institutes of Health grant R01AI124491 (to H.W.); the National Institutes of Health grant U19AI131135 (to L.G.); the Annenberg Foundation and FAST grants and a gift from J Sullivan (to A.E.G.); the American Lung Association (to M.B.G and M.R.F.); the British Heart Foundation Programme grant RG/16/4/32218 (to S.B.); a Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) fellowship (to C.J.); a Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) fellowship (to L.B.d.L.); and the National Institutes of Health training grant T32AI007245-31A1 (to M.L.) These authors contributed equally: Caroline Junqueira Program in Cellular and Molecular Medicine Department of Biological Chemistry and Molecular Pharmacology Institute for Medical Engineering and Science Dana-Farber Cancer Institute and Department of Cell Biology All of the authors contributed to preparing the manuscript The authors declare no competing interests reviewer(s) for their contribution to the peer review of this work Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Flow cytometry gating strategy for identifying lymphocytes and monocytes in Fig. 1a, b (a) and for identifying monocyte subpopulations in Fig. 1c (b) Monocyte subpopulations: CL - classical CD14hiCD16−; ITM - intermediate CD14hiCD16+; NCL - non-classical CD14loCD16+ COVID-19 patients (n = 4) and A549-ACE2 (n = 3) were analysed by flow cytometry (a HD monocytes were treated or not with LPS before analysis A549-ACE2 cells were used as positive control ****p<0.0001 relative to isotype (Iso) control antibody-stained LPS-activated HD monocytes (a,c) by one-way ANOVA with Tukey’s multiple comparisons test Data are representative of 2 independent experiments then stained 48 h later for nucleocapsid (N) or dsRNA (J2) and ASC and analysed by imaging flow cytometry virus was preincubated with indicated monoclonal antibodies (IgG1 isotype control mAb114 (Iso)) non-neutralizing anti-spike (C1A-H12 (H12)) or neutralizing anti-RBD (C1A-B12 (B12)) or with pooled HD or COVID-19 patient plasma that had been heat-inactivated (HI) or not Quantification of HD monocyte staining for N (a) (e) Shows the percentage of N+ cells that were also NG fluorescent A5490-ACE2 (n = 3) (f) or LPS-primed HD monocytes (n = 3) (g) were infected at the indicated MOI with icSARS-CoV-2-mNG (NG) a molecular clone of the Washington (WA) strain Infection was measured by N staining and flow cytometry ****p<0.0001 by one-way ANOVA with Tukey’s multiple comparisons test b) or purified HD neutrophils (n = 3) (c) were infected with icSARS-CoV-2-mNG (MOI then stained 48 h later for nucleocapsid (N) or analysed for NG fluorescence (c virus was preincubated with COVID-19 plasma and infection was carried out in the presence of indicated blocking or isotype (Iso) control antibodies (a–c) The monocyte and neutrophil infections in (b) and (c) were performed with cells isolated from the same HDs from HD (n = 3) and COVID-19 patients of mixed disease severity (n = 4) were stained for N and analysed by flow cytometry to assess in vivo infection Infection of LPS-primed HD monocytes (n = 3) with icSARS-CoV-2-mNG in the presence of pooled COVID-19 patient plasma Infection of A549-ACE2 (n = 3) with icSARS-CoV-2-mNG to verify the inhibitory activity of 10 µM Remdesivir Infection of A549-ACE2 (n = 3) and HD monocytes (n = 3) with icSARS-CoV-2-mNG in the presence of anti-ACE2 blocking antibody at different concentrations by one-way ANOVA with Tukey’s multiple comparisons test (a–c f) and two-tailed nonparametric unpaired multiple t-test (e) Data are representative of 2 replicate experiments Demographic and clinical information of the fresh PBMCs and plasma cohort hospitalization details and clinical information of the patients in the fresh PBMCs and plasma cohort Demographic and clinical information of the frozen plasma cohort hospitalization details and clinical information of the patients in the frozen plasma cohort Clinical information of COVID-19 lung autopsies Reagents and materials used for this manuscript chemicals and commercial kits (with sources and catalogue numbers) described in the Methods Download citation DOI: https://doi.org/10.1038/s41586-022-04702-4 Anyone you share the following link with will be able to read this content: a shareable link is not currently available for this article Signal Transduction and Targeted Therapy (2025) Sign up for the Nature Briefing newsletter — what matters in science France’s Mathis Margirier notched his third win of the season after a thrilling sprint finish against Mika Noodt at Challenge Vieux Boucau in the south of France with the German falling just yards before the line as he was being overtaken A stacked men’s field had seen fortunes ebb and flow throughout – with double Olympic champion Alistair Brownlee looking at various points as if he was in with a great chance of a first win since his encouraging return from ankle surgery only to fade into sixth late on as he jogged home as a precaution The podium was completed by 70.3 World Champion Rico Bogen It was slightly less dramatic in the women’s race as Germany’s Caroline Pohle denied Switzerland’s Imogen Simmonds a third win in the space of a fortnight Pohle had led out of the water before Simmonds pushed to the front on the bike but on the run it was Pohle who proved strongest to take the victory by 30 seconds Newly-crowned WTCS champion Dorian Coninx (FRA) was first out of the water after the wetsuit lake swim in 24:02 with Aaron Royle (AUS) Brownlee and Margirier all within a few seconds of him And on the predominantly flat single 90km bike loop through the famous Landes pine forests the conditions soon turned testing thanks to torrential rain and it was Brownlee who moved to the head of affairs at halfway But he was never able to get away and coming into T2 it effectively boiled down to a running race between the leading six which was now Noodt After just one of four loops around the Lac Marin de Port d’Albret six had become two as Noodt and Bogen opened up a gap of just over a minute but things would start to change dramatically as first Brownlee and then Margirier moved back into the mix As spectators cheered Noodt to victory on the red carpet Margirier was sprinting up behind him Noodt’s legs could not respond and he stumbled less than a metre before the line and Margirier flew through to take the win by a second Noodt said afterwards: “It’s insane – I gave everything but I lost control of my legs Meanwhile Brownlee revealed afterwards that he had eased off as a precaution after not feeling “quite right” Pohle set the pace in the swim and was first out of the water in 26:46 with Simmonds 37 seconds adrift in second and already a near two-minute gap to the rest But the catch came almost two thirds of the way into the 90km bike loop with Simmonds overtaking after 58km and quickly putting 40 seconds between them She was still ahead early on the run but the gap was coming down quickly and Pohle was back in front at the 5km mark and would never be caught with 30 seconds separating them on the line “I’m really proud of my achievement as I had a really difficult time after the World Championship with injury,” said Pohle at the finish line “I’m really happy as Imo was really strong and she pushed me to my best.” Metrics details Protein-based virus-like particles (P-VLPs) are commonly used to spatially organize antigens and enhance humoral immunity through multivalent antigen display P-VLPs are thymus-dependent antigens that are themselves immunogenic and can induce B cell responses that may neutralize the platform we investigate thymus-independent DNA origami as an alternative material for multivalent antigen display using the receptor binding domain (RBD) of the SARS-CoV-2 spike protein the primary target of neutralizing antibody responses Sequential immunization of mice with DNA-based VLPs (DNA-VLPs) elicits protective neutralizing antibodies to SARS-CoV-2 in a manner that depends on the valency of the antigen displayed and on T cell help class-switched antibodies against the DNA scaffold in contrast to P-VLPs that elicit strong B cell memory against both the target antigen and the scaffold DNA-VLPs enhance target antigen immunogenicity without generating scaffold-directed immunity and thereby offer an important alternative material for particulate vaccine design We find that sequential immunization with DNA-VLPs in mice boosts neutralizing and protective RBD-specific antibodies in a manner that is dependent on both antigen valency and T cell help We further show that in contrast to P-VLPs DNA-VLPs do not generate boostable antibodies against the scaffold this offers a proof-of-concept study that the antibody titer-enhancing benefits of multivalent protein antigen display can be decoupled from eliciting potent B cell responses against the scaffold platform itself a Recombinant RBD bearing an additional Cys residue at the C-terminus was expressed The C-terminal Cys was selectively labeled with an SMCC-azide linker and subsequently conjugated to DNA-VLPs displaying DBCO groups The icosahedral DNA origami objects of ~40 nm diameter displaying 1 b Agarose gel electrophoresis (AGE) shows the gel shift due to increasing RBD copy number as well as low polydispersity of the DNA-VLPs samples after purification An additional VLP bearing 5 copies of Cy5 was produced for ACE2 binding flow cytometry experiments Images are representative of n = 3 biological replicates c The coverage of the DNA-VLPs with RBD was quantified via Trp fluorescence Coverage values were determined from n = 3 biological replicates for DNA-VLP-1x and from n = 6 biological replicates for DNA-VLP-6x and DNA-VLP-30x d Dynamic light scattering (DLS) was used to assess the dispersity of functionalized VLP samples Representative histograms are shown from n = 3 biological replicates e Transmission electron micrographs (TEM) of DNA-VLP-30x were obtained by negative staining using 2% uranyl formate and validate the symmetric nanoscale organization of antigens Images are representative of n = 3 technical replicates Error bars and errors represent the standard error of the mean a An overview schematic of the in vitro activity assays and corresponding DNA-VLPs tested c ACE2-expressing HEK 293 cells were incubated with 200 nM RBD Binding was detected in flow cytometry experiments using PE-labeled CR3022 and a PE-labeled secondary antibody demonstrating preserved binding activity for chemically modified monomeric RBD-Cy5 compared to monomeric RBD Representative histograms are shown for ACE2 binding assays from n = 3 biological replicates and median fluorescent intensity (MFI) values were determined from n = 3 biological replicates e Incubation with Cy5-labeled DNA-VLP-Cy5-30x at 100 nM RBD revealed enhanced binding compared to monomeric RBD-Cy5 No unspecific binding for non-functionalized DNA-VLP-Cy5 was observed The brightness of Cy5-labeled DNA-VLP-Cy5-30x (5 Cy5 per 30 RBDs) and RBD-Cy5 (1 Cy5 per 1 RBD) were quantified experimentally and MFI values were corrected accordingly and MFI values were determined from n = 3 biological replicates g Ramos B cells expressing the BCRs CR3022 and B38 were incubated with α-IgM Ca2+ flux in response to RBD incubation was assayed using Fura Red Representative fluorescence intensity (FI) curves are shown from n = 3 biological replicates (top) Total Ca2+ flux was quantified via the normalized area under the curve (AUC) (bottom) Normalized AUC values were determined from n = 3 biological replicates Error bars represent the standard error of the mean Two-sided Welch’s t-test was performed at α = 0.05 for (c) One-way ANOVA was performed followed by Dunnett’s T3 multiple comparison test at α = 0.05 for (e Left: p = 0.0006 for Monomer-Cy5:DNA-VLP-Cy5-0x p = 0.0161 for Monomer-Cy5:DNA-VLP-Cy5-30x p = 0.0042 for DNA-VLP-Cy5-0x:DNA-VLP-Cy5-30x; Right: p < 0.0001 for Monomer-Cy5:DNA-VLP-Cy5-0x p = 0.0036 for Monomer-Cy5:DNA-VLP-Cy5-30x p = 0.0029 for DNA-VLP-Cy5-0x:DNA-VLP-Cy5-30x *p < 0.05; **p < 0.01; ***p < 0.001 When correcting for Cy5 brightness per RBD DNA-VLP-Cy5-30x displayed an approximately ten-fold increase in median fluorescence intensity compared with monomeric RBD-Cy5 likely due to avidity effects of multivalent DNA-VLPs binding to the cognate ACE2 receptors a Mice were sequentially immunized with monomeric RBD and DNA-VLPs of varying copy number b RBD-specific IgG endpoint dilutions revealed enhanced antibody responses for DNA-VLP-30x compared to both monomeric RBD and DNA-VLP-6x c Serum neutralization titers expressed as NT50 values against pseudoviruses modeling the ancestral Wuhan-1 strain d Serum neutralization titers expressed as NT50 against native Wuhan-1 SARS-CoV-2 e IgM and IgG titers of RBD-specific antibodies elicited in Tcra–/– and wild-type mice after sequential immunization with DNA-VLP-30x N = 5 female mice were used in each experimental group = not detectable) were not considered for statistical analysis One-way ANOVA was performed followed by Dunnett’s T3 multiple comparison test at α = 0.05 for (b Two-sided Welch’s t-test was performed at α = 0.05 for (d) Boost 1: p = 0.0021 for Monomer:DNA-VLP-30x p = 0.0022 for DNA-VLP-6x:DNA-VLP-30x; Boost 2: p = 0.0048 for DNA-VLP-6x:DNA-VLP-30x Boost 2: p = 0.0001 for Monomer:DNA-VLP-30x p = 0.0001 for DNA-VLP-6x:DNA-VLP-30x; Boost 2 Normalized: p = 0.0005 for Monomer:DNA-VLP-30x; p = 0.0002 for DNA-VLP-6x:DNA-VLP-30x IgG boosting following sequential immunization with DNA-VLPs was due to TD a Mice were sequentially immunized with monomeric RBD b RBD-specific and scaffold-specific IgG endpoint dilutions for the P-VLP immunization c RBD-specific and scaffold-specific IgG endpoint dilutions for the DNA-VLP immunization and dilution curves for Boost 2 of the DNA-specific IgG ELISA The DNA-specific IgG control was diluted from 10 µg/ml N = 5 male mice were used in each experimental group RBD-specific IgG titers: p = 0.0199 for Prime:Boost 1 p = 0.0209 for Boost1:Boost 2; Ferritin-specific IgG titers: p = 0.0036 for Prime:Boost 1 Our findings suggest that a TI scaffold can mitigate this issue of anti-vector antibodies by ensuring: (1) valency-dependent antibody boosting against the scaffolded protein antigen within the TD pathway; and (2) confinement of scaffold-directed immunity to the TI pathway The research conducted complies with ethical regulations and biosafety approved by institutional committees at Mass General Brigham (MGB Institutional Biosafety Committee 2018B000030; MGB Institutional Animal Care and Use Committee protocol #2014N000252); MIT Institutional Biosafety Committee (protocol #BRR673); Washington University Institutional Biosafety Committee (protocol #14366); Washington University Animal Care and Use Committee (protocol #21-0246) This research does not include human participants Endotoxin levels were measured using ToxinSensor Gel Clot Endotoxin Assay Kits Purity of the scaffold was analyzed by agarose gel electrophoresis (AGE) (1.6% agarose For the assembly of DNA-VLP-Cy5 and DNA-VLP-Cy5-30x-DBCO Cy5-modified oligonucleotide staples were synthesized 10x excess of Cy5-Azide was added to 50 µM DBCO-modified oligonucleotide staple in PBS with 10% DMF and incubated overnight at room temperature Excess dye was removed using NAP-5 columns prior to purification of Cy5-modified oligonucleotide staples via reversed-phase HPLC (BEH-C18 column; 30 °C; 0.1 M triethylammonium acetate in water:acetonitrile gradient) The codon-optimized gene for the expression of the RBD of SARS-CoV-2 (GenBank ID = MN975262.1; residues 319-529) was cloned into a pVRC vector containing a C-terminal HRV C3 protease cleavage site followed by 8x His and SBP tags An additional C-terminal Cys residue was inserted using QuickChange Mutagenesis following the manufacturer’s protocol and mutagenesis was confirmed by next-generation sequencing (Azenta) to afford RBD-Cys HEK Expi293F cells were transiently transfected with the RBD plasmid using Expifectamine following the manufacturer’s protocol supernatants were harvested by centrifugation at 4000 g at room temperature for 5 min and the RBD-Cys was purified into PBS by affinity chromatography using TALON cobalt resin followed by size-exclusion chromatography using Superdex 200 Increase columns and stored at 4 °C for less than 7 days Antigen modification with the azide linker was adapted from published protocols48 100 µM RBD-Cys was incubated with 10x excess of TCEP in PBS for 30 min at room temperature and subsequently purified into PBS with 10 mM EDTA using Zeba spin columns (7 kDa) Incubation for ~6 h at room temperature allowed for the reoxidation of disulfides prior to the addition of the azide linker as monitored by Ellman’s assay The azide linker was assembled by incubation of 60 mM SMCC with 1.1x excess of amino-TEG-azide for 1 h at room temperature 10x excess of crude azide linker was added to reduced antigen and the reaction mixture was incubated overnight at room temperature to afford RBD-Az RBD-Az was purified into PBS using Amicon Ultra centrifugal filters (10 kDa 5000 g) followed by size-exclusion chromatography using Superdex 200 Increase columns and stored at 4 °C for less than 7 days 50 µM RDB-Az was incubated with 5x excess of DBCO-Cy5 in PBS for 30 min at room temperature and subsequently purified into PBS using Amicon Ultra centrifugal filters (10 kDa RBD concentrations and dye labeling efficiency were determined by absorbance measurements at 280 nm (ε = 39400 1/(M∙cm) and MW = 31 kDa) and 650 nm (ε = 250000 1/(M∙cm)) Uranyl formate staining of DNA-VLP samples was adapted from an existing protocol97 and 5 µl of the solution were immediately deposited onto glow-discharged electron microscopy grids the solution was removed by blotting with filter paper and the grids were washed with 5 µl of freshly prepared 2% uranyl formate with 5 mM NaOH After removal of the washing solution by blotting 15 µl of the uranyl formate solution was added incubated for 30 s and the removed by blotting the grids were dried in vacuo and transmission electron microscopy (TEM FEI Tecnai G2 Spirit Twin) was conducted at 120 keV Biolayer interferometry binding experiments were performed after immobilization of CR3022 and B38 Fab at 0.1 mg/ml on FAB2G sensors (BLItz Wild-type RBD and RBD-Az served as analytes at 10 µM in the manufacturer’s Kinetics Buffer ACE2 expressing HEK 293T cells (generously provided by Nir Hacohen and Michael Farzan Massachusetts General Hospital and The Scripps Research Institute) were harvested and washed with PBS with 2% FBS 200,000 cells per well were transferred to 96-well cell culture plates and 100 µl of wild-type RBD and RBD-Cy5 at concentrations corresponding to 200 nM RBD in PBS were added cells were washed twice with PBS with 2% FBS and stained with 50 µl at 200 nM of the anti-RBD antibody CR3022 for 30 min at room temperature following pre-complexation with goat anti-human-PE at 200x excess The suspension was protected from light and incubated for 30 min on ice washed twice with PBS with 2% FBS and resuspended in 100 µl PBS with 2% FBS cell binding was also detected via direct Cy5 fluorescence After incubation with RBD-Cy5 or DNA-VLPs at concentrations corresponding to 100 nM RBD (DNA-VLP-Cy5 concentration was equivalent to DNA-VLP-Cy5-30x) no staining was performed and cells were directly resuspended in 100 µl PBS with 2% FBS Cell binding was analyzed by flow cytometry (S1000Exi Flow Cytometer Strategim) and data processing was conducted using FlowJo (BD Biosciences Cy5 fluorescence intensities obtained by flow cytometry were corrected according the relative brightness of RBD-Cy5 and DNA-VLP-Cy5-30x as quantified by fluorescence spectroscopy in PBS BCR-expressing Ramos B cells were FACS-sorted for IgM and κ light chain expression (SH800S Cell Sorter Sorted cells were expanded in RPMI supplemented with 15% FBS and 1x penicillin-streptomycin-glutamine and 1,000,000 cells were harvested and resuspended RPMI with Fura Red solution following the manufacturer’s protocol cells were harvested and resuspended in 500 µl RPMI medium prior to detection of BCR signaling by flow cytometry at 637 nm Following 30 s of baseline data acquisition or P-VLP-24x at concentrations corresponding to 30 nM RBD were added before continuing data acquisition for an additional 270 s The concentration of DNA-VLP-0x corresponded to that of DNA-VLP-1x Goat anti-human IgM at 10 µg/ml served as a positive control Maximum total Ca2+ flux was measured after addition of 10 µg/ml ionomycin Fluorescence traces were processed as follows: For each trace the average fluorescence of the 30 s baseline data acquisition was subtracted the fluorescence traces were normalized to the Ca2+ flux induced by ionomycin to obtain relative Ca2+ flux traces The total Ca2+ flux was quantified by integration to obtain the normalized area under the curve (AUC) and 6 and blood draws occurred 2 weeks after each immunization The animals were maintained within the Ragon Institute’s HPPF barrier facility and the experiments were conducted with IACUC approval (MGH protocol 2014N000252) The light cycles in the animal room were set on a 12 h light cycle [7AM-7PM (ON) 7PM-7AM (OFF)] The temperature range for the room was 68–73 °F and the humidity index was from 30–70% the animals were euthanized by CO2 inhalation (30% of the chamber volume/min) except that sheep anti-human IgG-HRP (GE Healthcare) was used as the secondary antibody Calf-thymus DNA was reconstituted in water 50 µg/ml and used to coat 96-well MaxiSorp plates overnight at 4 °C blocked with 1% casein buffer for 2 h at room temperature and subsequently washed with PBS Mouse sera (1:30) or mouse anti-dsDNA antibody (1:100) were diluted in blocking buffer transferred to the plates and incubated for 2 h at room temperature The plates were washed with PBS with 0.2% Tween-20 and incubated with 1:5000 dilution goat anti-mouse-IgG-HRP antibody (BioRad) for 1 h at room temperature Following washing with PBS with 0.2% Tween-20 the plates were incubated for 3 min and the reaction was stopped using sulfuric acid DNA-specific IgG titers were determined by absorbance measurements at 450 nm Endpoint dilutions were calculated using an absorbance cut-off of 0.05 as determined from the limit of detection determined for PBS-incubated wells (Graphpad Prism v.9.1 SARS-CoV-2 neutralization was assessed using pseudotyped lentivirus particles expressing S glycoprotein trimer as previously described71 pseudovirus corresponding to the ancenstral Wuhan-1 strain were produced by transient transfection of HEK 293T cells The titers of viral supernatants were determined via flow cytometry with ACE2-expressing HEK 293T cells and via the HIV-1 p24CA antigen capture assay (Leidos Biomedical Research) Assays were performed in 384-well plates using a fluorescence plate reader (Tecan Fluent Automated Workstation) Mouse sera (or CR3022 and B38 mAb standards) were serially 3x diluted in 20 µl followed by the addition of 20 µl of pseudovirus containing 250 IFU and incubated for 1 h at room temperature Next 10,000 ACE2-expressing HEK 293T cells are were added per well and incubated for 60 to 72 h at 37 °C cells were lysed and incubated on a shaker for 5 min at room temperature before measuring luciferase expression (Molecular Devices SpectraMax L) Relative neutralization for each serum dilution was calculated after subtracting background luminescence and dividing by the luminescence in absence of sera NT50 vales were derived by fitting the Dose-Response equations to serum dilution curves SARS-CoV-2 neutralization was also assessed using authentic virus as previously described73 A549-hACE2 cells were detached using Trypsin-EDTA (ThermoFisher Scientific) and seeded at 40,000 cells per well in 96-well plates 16–20 h before infection the cell culture supernatant was removed and 75 µL of D2+ media was added (2% FBS instead of 10%) Mouse sera were diluted in D+ media (no FBS) in 3-fold serial dilutions mixed 1:1 (v/v) with SARS-CoV-2 diluted at 40,000 pfu/ml and incubated at 37 °C and 5% CO2 for 1 h 25 µl of the sera-virus solutions were added in triplicate wells for each condition for a final multiplicity of infection of 0.01 (virus to cell ratio) The 96-well plates were centrifuged for 30 min at 2000 g at 37 °C and then incubated at 37 °C and 5% CO2 for 48 h Each plate included a no infection control a no treatment control for maximum infection as well serially diluted B38 mAb as a positive control the cell culture supernatant was discarded the cells were washed with PBS (Corning) then harvested using TrypLE (Life Technologies) and flow cytometry buffer (2% FBS in PBS) then stained with live/dead fixable blue stain (ThermoFisher Scientific) for 30 min at 4 °C the cells were fixed using 4% paraformaldehyde (Santa Cruz) for 30 min at 4 °C The fixed cells were removed from the BSL3 laboratory and prepared for intracellular staining using Perm/Wash buffer (BD Biosciences) The permeabilized cells were stained with mouse anti-SARS-CoV-2 Nucleocapsid antibody (Biolegend) for 30 min at 4 °C then washed and stained with secondary antibody labeled with Pe-Cy7 (Biolegend) for 30 min at 4 °C the cells were washed and resuspended in flow cytometry buffer Flow cytometry was performed on a BD Symphony (BD Biosciences) FCS files were analyzed using FlowJo software (v.10 Additional data analysis was performed using GraphPad Prism (v.9.1 GraphPad Software Inc) to fit curves for the neutralization data a TaqMan assay was designed to target a highly conserved region of the N gene: Probe: /56-FAM/TCAAGGAAC/ZEN/AACATTGCCAA/3IABkFQ/ This region was included in an RNA standard to allow for copy number determination down to 10 copies per reaction The reaction mixture contained final concentrations of primers and probe of 500 and 100 nM Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article Eliciting B cell immunity against infectious diseases using nanovaccines Protein-based antigen presentation platforms for nanoparticle vaccines Shaping humoral immunity to vaccines through antigen-displaying nanoparticles Nanoparticle size influences antigen retention and presentation in lymph node follicles for humoral immunity Targeted modulation of immune cells and tissues using engineered biomaterials B cells acquire antigen from target cells after synapse formation Precursor frequency and affinity determine B cell competitive fitness in germinal centers tested with germline-targeting HIV vaccine immunogens Induction of potent neutralizing antibody responses by a designed protein nanoparticle vaccine for respiratory syncytial virus Multifaceted effects of antigen valency on B cell response composition and differentiation in vivo protein nanoparticles presenting the receptor binding domain and stabilized spike as SARS-CoV-2 vaccine candidates how and where of antigen presentation to B cells Antigen receptor function in the context of the nanoscale organization of the B cell membrane Molecular determinants of immunogenicity: the immunon model of immune response IgG B memory cell subpopulations: differences in susceptibility to stimulation by TI-1 and TI-2 antigens T-independent type II immune responses generate memory B cells Innate B cell helpers reveal novel types of antibody responses Marginal zone B cells: virtues of innate-like antibody-producing lymphocytes T follicular helper cell biology: a decade of discovery and diseases B cell responses: cell interaction dynamics and decisions Quadrivalent influenza nanoparticle vaccines induce broad protection Self-assembling influenza nanoparticle vaccines elicit broadly neutralizing H1N1 antibodies Rational design of an epstein-barr virus vaccine targeting the receptor-binding site Mosaic nanoparticle display of diverse influenza virus hemagglutinins elicits broad B cell responses Particulate array of well-ordered HIV clade C Env trimers elicits neutralizing antibodies that display a unique V2 cap approach A virus-like particle vaccine for epidemic Chikungunya virus protects nonhuman primates against infection Adjuvanting a subunit COVID-19 vaccine to induce protective immunity Mosaic nanoparticles elicit cross-reactive immune responses to zoonotic coronaviruses in mice Mosaic RBD nanoparticles protect against challenge by diverse sarbecoviruses in animal models Engineered SARS-CoV-2 receptor binding domain improves manufacturability in yeast and immunogenicity in mice Efficacy and breadth of adjuvanted SARS-CoV-2 receptor-binding domain nanoparticle vaccine in macaques SARS-CoV-2 ferritin nanoparticle vaccines elicit broad SARS coronavirus immunogenicity Hemagglutinin-stem nanoparticles generate heterosubtypic influenza protection Priming a broadly neutralizing antibody response to HIV-1 using a germline-targeting immunogen Immunogenic display of diverse peptides on virus-like particles of RNA phage MS2 Enhancing humoral responses to a malaria antigen with nanoparticle vaccines that expand Tfh cells and promote germinal center induction Elicitation of potent neutralizing antibody responses by designed protein nanoparticle vaccines for SARS-CoV-2 Rationally designed immunogens enable immune focusing following SARS-CoV-2 spike imprinting Antigen- and scaffold-specific antibody responses to protein nanoparticle immunogens Carrier-priming leads to hapten-specific suppression Virus-like display of a neo-self antigen reverses B cell anergy in a B cell receptor transgenic mouse model Immune imprinting and SARS-CoV-2 vaccine design Development of novel adenoviral vectors to overcome challenges observed with HAdV-5-based constructs Merck Ad5/HIV induces broad innate immune activation that predicts CD8(+) T-cell responses but is attenuated by preexisting Ad5 immunity Folding DNA to create nanoscale shapes and patterns Designer nanoscale DNA assemblies programmed from the top down In situ covalent functionalization of DNA origami virus-like particles Rapid prototyping of arbitrary 2D and 3D wireframe DNA origami Programming structured DNA assemblies to probe biophysical processes Functionalizing DNA origami to investigate and interact with biological systems Binding to nanopatterned antigens is dominated by the 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after immunization Controlling nuclease degradation of wireframe DNA origami with minor groove binders A DNA nanodevice-based vaccine for cancer immunotherapy Innate immune stimulation using 3D wireframe DNA origami Spatially controlled activation of toll-like receptor 9 with DNA-based nanomaterials Oligolysine-based coating protects DNA nanostructures from low-salt denaturation and nuclease degradation Gene synthesis machines: DNA chemistry and its uses Allelic polymorphism controls autoreactivity and vaccine elicitation of human broadly neutralizing antibodies against influenza virus Naive human B cells engage the receptor binding domain of SARS-CoV-2 Resistance of SARS-CoV-2 variants to neutralization by monoclonal and serum-derived polyclonal antibodies A SARS-CoV-2 infection model in mice demonstrates protection by neutralizing antibodies Download references was additionally supported by the Feodor Lynen Fellowship of the Alexander von Humboldt Foundation was additionally supported by the National Science Foundation under a Graduate Research Fellowship 4000189657 was additionally supported by the National Science Foundation under a Graduate Research Fellowship 4000168384 acknowledge MIT.nano for the use of core characterization facilities and acknowledge support from core center grants P30-ES002109 was supported by NIGMS T32GM007753 and F30 AI160908 was supported by NIH R01 AI146779 and a Massachusetts Consortium on Pathogenesis Readiness (MassCPR) grant DP2DA040254 and CDC subcontract 200-2016-91773-T.O.2 and a Massachusetts Consortium on Pathogenesis Readiness (MassCPR) grant SARS-CoV-2 virus work was performed in part at the Ragon Institute’s Biosafety Level 3 facility which is supported by the NIH-funded Harvard University Center for AIDS Research (P30 AI060354) and the Massachusetts Consortium on Pathogen Readiness (MassCPR) The authors are also grateful to Vintus Okonkwo and Faez Amokrane Nait Mohamed in the Lingwood lab for technical assistance and maintenance of B cell lines and Benjamin Clancy for technical assistance on DNA origami materials production These authors contributed equally: Eike-Christian Wamhoff Ragon Institute of Massachusetts General Hospital Massachusetts Institute of Technology and Harvard University Koch Institute for Integrative Cancer Research Harvard Medical School Initiative for RNA Medicine The Massachusetts Institute of Technology has filed a patent (US application number 16/752,394) covering the use of DNA origami as a vaccine platform on behalf of the co-inventors (E.-C.W The Diamond laboratory has received unrelated funding support in sponsored research agreements from Emergent BioSolutions reports SAB membership for Flagship Labs 72 is co-founder and SAB member of Kano Therapeutics The remaining authors declare no competing interests Nature Communications thanks Abhisek Dwivedy and the other Download citation DOI: https://doi.org/10.1038/s41467-024-44869-0 Sign up for the Nature Briefing: Microbiology newsletter — what matters in microbiology research THE Challenge Vieux Boucau triathlon ended in heartbreak for German athlete Mika Noodt The 23-year-old led for almost the entire running stage before he was pipped to the finish line by Frenchman Mathis Margirier with less than a metre to go Noodt initiated a final push as he powered down the home straight With just a few steps to go he looked over his shoulder to find Margirier hot on his heels the German "lost control" of his legs and fell to the floor His rival then clinched the win in dramatic fashion while the crowd looked on in shock "I’ve never experienced this – he was too fast, but that’s racing – I gave it everything I had!" Meanwhile, Margirier revealed he didn't think he would be able to get the win until the final stage of the race. He said: "It was really hard today. I wasn’t feeling good on the bike so in the middle of the bike, I was just waiting for the finish. "The first lap of the run was so hard but I could sprint and he couldn’t so I finished first!" Olympic gold medallist Alistair Brownlee also took part in the inaugural Challenge Vieux Boucau Our journalists strive for accuracy but on occasion we make mistakes. For further details of our complaints policy and to make a complaint please click this link: thesun.co.uk/editorial-complaints/ The finishing straight at triathlon’s inaugural Challenge Vieux Boucau saw things turn from ecstasy to agony for Mika Noodt as victory was snatched from his grasp while any chance of a win for Alistair Brownlee also evaporated in the closing stages Despite being up against a super-strong field for almost the entire run it appeared that first place was going to Noodt even though 70.3 Worlds winner Rico Bogen was not making it easy for him first Brownlee and then Mathis Margirier were gaining fast and it was the latter who would prove the big danger As spectators cheered Noodt to victory on the red carpet Margirier was sprinting up behind him and Noodt’s legs could not respond – meaning he stumbled less than a metre before the line and Margirier flew through to take the win I gave everything but I lost control of my legs,” said Noodt afterwards “I’ve never experienced this – he was too fast But that’s racing – I gave it everything I had!” Margirier revealed that for most of the day he thought the win would be beyond him “It was really hard today,” he explained “I wasn’t feeling good on the bike so in the middle of the bike “The first lap of the run was so hard but I could sprint and he couldn’t so I finished first!” Meanwhile Brownlee revealed afterwards that he had eased back in the closing stages after not feeling “quite right” Writing on social media he said: “Another tough race day with tough weather and even tougher competition at Challenge Vieux Boucau “Wasn’t feeling my best but was enjoying being out on the course and closing in on the lead in the late stages of the run Didn’t feel quite right on the last lap so decided to jog it home to not put myself at risk for future races after the year I’ve had “Hats off to Mathis and Mika for an awesome sprint finish.”