key: cord-297974-sduz0j35
authors: Bokelmann, L.; Nickel, O.; Maricic, T.; Paabo, S.; Meyer, M.; Borte, S.; Riesenberg, S.
title: Rapid, reliable, and cheap point-of-care bulk testing for SARS-CoV-2 by combining hybridization capture with improved colorimetric LAMP (Cap-iLAMP)
date: 2020-08-06
journal: nan
DOI: 10.1101/2020.08.04.20168617
sha: 
doc_id: 297974
cord_uid: sduz0j35

Efforts to contain the spread of SARS-CoV-2 have spurred the need for reliable, rapid, and cost-effective diagnostic methods which can easily be applied to large numbers of people. However, current standard protocols for the detection of viral nucleic acids while sensitive, require a high level of automation, sophisticated laboratory equipment and trained personnel to achieve throughputs that allow whole communities to be tested on a regular basis. Here we present Cap-iLAMP (capture and improved loop-mediated isothermal amplification). This method combines a hybridization capture-based RNA extraction of non-invasive gargle lavage samples to concentrate samples and remove inhibitors with an improved colorimetric RT-LAMP assay and smartphone-based color scoring. Cap-iLAMP is compatible with point-of-care testing and enables the detection of SARS-CoV-2 positive samples in less than one hour. In contrast to direct addition of the sample to improved LAMP (iLAMP), Cap-iLAMP does not result in false positives and single infected samples can be detected in a pool among 25 uninfected samples, thus reducing the technical cost per test to ~1 Euro per individual.

The recent global outbreak of coronavirus disease 2019 has led governments to take drastic measures to contain the spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Most affected countries resorted to more or less stringent lockdown measures including restrictions on travel, public gatherings and the closing of institutions such as schools, kindergartens and universities. These restrictions and closings have usually been implemented broadly, affecting the lives of infected and uninfected people alike, as no reliable and economical mass testing approach for decentralized point-of-care identification of infected individuals exists.

Reverse transcription followed by quantitative PCR (RT-qPCR) is the most widely used method to detect RNA viruses such as SARS-CoV-2. However, its need for trained personnel and expensive instrumentation and global shortages of resources for RNA purification has spurred search for viable alternatives. Loop-mediated isothermal amplification (LAMP) can rapidly amplify small target nucleic acid sequences under isothermal conditions (Notomi et al. 2000) and has been applied for molecular diagnostics (Wong et al. 2018) . The reaction requires four to six primers and produces concatemers of double-stranded amplification products. These can be detected directly, using intercalating dyes (e.g. SYBR green, SYTO-dyes), its reaction with triphenylmethane dye precursors and acid hydrolysis (Miyamoto et al. 2015; Trinh and Lee 2019), or using cleavage with CRISPR enzymes coupled with lateral flow color detection of the cleavage product (Broughton et al. 2020 ). However, these methods require opening of the tube after the reaction, thus posing the threat of cross-contaminating future reactions with the amplified product. Amplification can also be detected indirectly by hydroxynaptholblue or phenol red based detection of the release of protons and/or pyrophosphate generated during DNA synthesis (Tanner et al. 2015) . Recently, a number of studies explored ways to detect SARS-CoV-2 RNA using RT-LAMP (Lamb et al. 2020; Yang et al. 2020; Zhang et al. 2020) but they required time consuming RNA isolation steps before the reaction. There have also been attempts to add sample directly into the reaction without prior purification (Buck et al. 2020; Dao Thi et al. 2020 ). However, it was noted that the pH of nasopharyngeal swab samples often varies and can adversely affect readouts.

Here we describe a method to detect SARS-CoV-2 RNA of a single infected individual within a bulk sample comprised of up to 26 individual patient samples by combining a hybridizationcapture-based RNA extraction approach with smartphone app-assisted colorimetric detection of RT-LAMP products, a procedure that can be performed in less than one hour ( Figure 1A) .

We compared the sensitivity of the method to standard extraction RT-qPCR protocols in a diagnostic lab and validate its performance on 287 gargle lavage samples from a hospital, a nursing home previously affected by COVID-19, and round robin samples from a reference institution of the German Medical Association.

We evaluated three published RT-LAMP primer combinations targeting either the Orf1a gene or the N gene of the SARS-CoV-2 genome (Lamb et al. 2020; Zhang et al. 2020 ) using a dilution series of synthetic viral RNA (Twist Biosciences, San Francisco, CA, USA) and chose the two most sensitive primer sets (CV1-6 and CV15-20, Supp. Table 1) Table 1 ). Using both primer combinations, we detected 500 synthetic viral RNA copies after 25-30 minutes incubation at 65°C as measured by fluorescence real time RT-LAMP (Supp. Figure 1A and C).

Combining the primers for the Orf1a gene and the N gene did not increase sensitivity (Supp. Figure 1D ).

Amplification in LAMP reactions is often detected colorimetrically by a pH sensitive dye that changes color when extensive DNA synthesis lowers the pH of the reaction (Tanner et al. 2015) . As noted in a previous study (Dao Thi et al. 2020) , biological samples such as nasopharyngeal swab eluates may change the pH when added to the LAMP reaction directly, leading to false positive results. We found that nasopharyngeal swab eluates tend to be more acidic than gargle lavage samples and that adding gargle lavage directly to a LAMP reaction at a final concentration of 5% leads to false positive results in 4.7% of cases even before the isothermal incubation (Supp. Figure 2A and B).

It would therefore be preferable to detect the amplification product directly rather than indirectly by a pH change. To achieve this, we deposited a drop of 0.5 microliters of a 10,000x concentrated solution of the dye SYBR Green I to the cap of the tube before the reaction is initiated. Shaking the tube after the isothermal amplification dissolves SYBR Green I, stops the reaction and allows visual detection of SARS-CoV-2 via a color change from orange/red to intense yellow ( Figure 1C ). The color of the reaction can be objectively quantified as a single numerical hue value, that is insensitive to light intensity changes and can be derived from the red, green, blue (RGB) color model (Cappi et al. 2015; Yin et al. 2019) , by using freely available 'camera color picker' smartphone apps. We used the 'Palette Cam' app (Alexander Mathers, App Store) for extracting RGB values before conversion to hue. An additional advantage is that this detection strategy allows us to include acidifying enhancing enzymes in the LAMP reaction, namely Tte UvrD helicase, which prevents unspecific late amplification of artefacts All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 6, 2020. . https://doi.org/10.1101/2020.08.04.20168617 doi: medRxiv preprint induced by primer interactions (Supp. Figure 3A and B) and thermostable inorganic pyrophosphatase which increases reaction speed (Miyamoto et al. 2015) .

However, addition of patient gargle lavage directly to this improved LAMP (iLAMP) reaction promoted false positives (13.5 %) (Supp. Figure 4A ). Presumably, this unspecific amplification is due to DNA from the oral microbiome, food or host cells as it can be prevented by prior λ exonuclease treatment that preferentially digests 5'-phosphorylated DNA leaving nonphosphorylated primers and iLAMP product intact (Supp. Figure 4B ). Because, gargle lavages from known infected patients did result in false negatives for all seven samples we employed an initial Quick extract (Lucigen, Middleton, WI, USA) lysis (Ladha et al. 2020) to release more viral RNA from the capsid. Even when these optimized reaction conditions were used, reactions resulted in one false negative out of the seven gargle lavages from infected patients and the single SARS-CoV-2 negative gargle lavage sample was false positive (Supp. Figure   4B ). We therefore employed a rapid (15min) bead-capture enrichment purification akin to mRNA isolation, using two oligonucleotides flanking the RT-LAMP target sites ( Figure 1B , Supp. Table 1 ) immobilized on paramagnetic beads. This step eliminates non-target nucleic acids and other unwanted components in the biological samples but also concentrates the viral RNA. Comparing the Ct-values of RT-qPCR targeting the E gene (Corman et al. 2020 ) after silica-based RNA-extraction with hybridization capture targeting the E gene for the same volume of gargle lavage suggests a capture efficiency of roughly 5% ( Figure 1D ). To allow comparison to RT-qPCR we captured viral RNA with a biotinylated probe for the E gene and not for the Orf1a and N gene as used for RT-iLAMP. When RNA is concentrated from 500µl gargle lavage to 25µl final volume and 10µl input volume is used in iLAMP, this results in a detection limit of 5-25 viral genome copies per µl of sample before capture.

The reagents required for the iLAMP reaction can be pre-mixed and freeze-thawed at least twice. Cap-iLAMP could be performed at point-of-care as no bulky equipment and only pipettes, a thermoblock, and a magnetic rack are needed ( Figure 1E ).

We used Cap-iLAMP to test 287 gargle lavage samples from hospital patients and elderly nursing home inhabitants and employees either individually or in pools. These samples had previously been tested by a RT-qPCR assay targeting the SARS-CoV-2 E gene (Maricic et al. 2020 ) and showed either no amplification or Ct values ranging from 24.6 to 32.7 ( Figure 2A ).

Cap-iLAMP targeting the SARS-CoV-2 Orf1a gene or the N gene results in an orange/red and intense yellow color for SARS-CoV-2 RNA negative and positive samples, respectively ( Figure   2B ). Of the 12 samples which we tested individually ( Figure 2C) , six had been previously tested All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 6, 2020. . https://doi.org/10.1101/2020.08.04.20168617 doi: medRxiv preprint positive in the RT-qPCR assay. Scoring the color of the iLAMP reactions using a smartphone 'camera color picker' app shows that hues for SARS-CoV-2 negative and positive samples are clearly separated, below 26° for the former and above 37° for the latter, respectively. All six negative samples were correctly identified as negative in the Cap-iLAMP assays targeting the Orf1a and the N gene. Of the six samples that were SARS-CoV-2 positive in the RT-qPCR assay, four were positive in the Cap-iLAMP assay while the remaining two were false negative.

When one twentieth of input volume were used they were positive, suggesting that some residual inhibition originating either from the biological sample or from lysis/binding buffer carryover exists in these extracts.

To investigate whether it is possible to detect single infected individuals in pools of gargle lavage samples, we created eleven pools of 25 patient samples each, all of which had been tested negative in RT-qPCR assay and in the Cap-iLAMP assays for the Orf1a and the N gene ( Figure 2D ). In order to determine if components of the pooled gargle lavage still inhibit the RT-LAMP reaction after capture, we took subsamples of the 10 negative pools and added 1000 copies/µl of artificial viral RNA before Cap-iLAMP. All 10 pools were positive in both Cap-iLAMP assays, showing that there was no substantial inhibition in these extracts after capture.

To investigate whether it is possible to detect a single infectious individual within a pool, we added different single positive patient samples (Ct < 26) to three pools of healthy individuals so that 1/26 (3.8%) of the final volume was composed of the infected sample. In all three cases, the Cap-iLAMP assays targeting the Orf1a and the N gene were positive, demonstrating that a single infectious individual can be detected in a pool of 26 samples. The two SARS-CoV-2 positive samples previously found to be inhibited when tested individually ( Figure 2C ) were correctly identified as positive when pooled ( Figure 2D ), potentially because inhibition is rare and is diluted out in the pool. An additional wash step after hybridization capture should thus be applied for individual sample diagnostic testing.

All tested gargle lavages from single healthy individuals (n=6) and 11 pools of 25 healthy individuals (n=275) correctly tested negative for both the Orf1a gene and N gene in the Cap-iLAMP assay ( Figure 2C and E), indicating that false positive results which were sometimes observed when samples are added directly into the iLAMP reaction (Supp. Figure 4A perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 6, 2020. . https://doi.org/10.1101/2020.08.04.20168617 doi: medRxiv preprint amounts of SARS-CoV-2 (ring 59, 61, 63 and 64), different coronaviruses (ring 60 HCoV OC43 and ring 65 HCoV 229E) or no virus (ring 62) and had previously been tested via RT-qPCR (Maricic et al. 2020 ) ( Figure 3A ). To prevent sporadic inhibition as observed for some gargle lavage samples, the first wash step after capture hybridization was performed twice. As shown in Figure 3B , all four SARS-CoV-2-containing samples were correctly and consistently identified by both the Orf1a and N gene assays while all samples devoid of virus or containing a different coronavirus were correctly classified as being negative.

We have established Cap-iLAMP as a reliable and rapid diagnostic test for SARS-CoV-2. Our method overcomes problems associated with standard RT-LAMP pH-dependent colorimetric detection which is prone to false positives due to pH-variability in gargle lavages and off-target amplification. Gargle lavage cause no discomfort and is thus more suitable than pharyngeal and nasal swabs for frequent and repeated testing of apparently healthy individuals (Saito et al. 2020) . They have the additional advantage that they do not expose the person collecting samples to any risk of infection.

In contrast to RT-qPCR approaches, no complicated equipment such as qPCR machines is required. Removal of inhibitors and enrichment of target nucleic acids by on-bead capture, combined with a double assay of two SARS-CoV-2-specific target sites and control reactions to detect inhibitor carryover and contaminated reagents ensure accuracy. As quality controls we recommend dedicated water negative controls and sample-specific inhibition/positive controls containing sample and synthetic viral RNA for each the Orf1a and the N gene assay.

A scheme for evaluating the results is shown in Supp. Table 2 .

Due to the removal of non-target nucleic acids and the inclusion of LAMP enhancing enzymes in Cap-iLAMP, we did not detect false positive results, which were often reported in previous LAMP-based detection methods (Teng et al. 2007; Abbasi et al. 2016; Hardinge and Murray 2019) . The inclusion of dye in the cap of the tube allows to keep the tube closed for detection after amplification thus preventing the cross-contamination of future assays. We quantified color as a single numeric hue value using a freely available smartphone app, which allows objective point-of-care detection without the ambiguity of an individual's perception of color.

Yin et al. (Yin et al. 2019 ) already described application of hue for hydroxynaptholblue color scoring that is in need of a custom-made 'smart cup' reaction tube holder as well as an app that is not freely available. Consequently, Cap-iLAMP outperforms other published SARS-All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 6, 2020. . https://doi.org/10.1101/2020.08.04.20168617 doi: medRxiv preprint CoV-2 detection methods (Broughton et al. 2020; Buck et al. 2020; Corman et al. 2020; Dao Thi et al. 2020 ) in some key aspects as shown in Table 1 .

The Cap-iLAMP assay passed an official round robin test used to evaluate testing accuracy across diagnostic laboratories. We show that it is possible to detect a single infected individual in a pool of 26 samples, opening the prospect of rapid screening of large numbers of people in public decentralized settings such as in schools, hospitals, airports, prisons, retirement homes or border crossings. This also allows daily testing of people at risk or working in critical infrastructure.

The Cap-iLAMP method can be readily applied to numerous human respiratory pathogens, plant pathogens, animal pathogens, food-borne diseases, viruses, protozoan parasites, and fungi, for which primer combinations have been developed (Wong et al. 2018 ). These include the eight diseases prioritized by the WHO (as of July 2020) that pose the greatest public health risk due to their epidemic potential and/or whether there is no or insufficient countermeasures (Kurosaki et al. 2010; Fukuma et al. 2011; Osman et al. 2013; Oloniniyi et al. 2017; Huang et al. 2018; Ma et al. 2019) . For testing in remote areas or developing countries master mixes for iLAMP could potentially be lyophilized for transportation or long term storage, as shown previously (Chen et al. 2016; Carter et al. 2017) .

Sampling of gargle lavages was done as described previously by our group (Maricic et al. 2020 ). All individuals included in this study were asked for their voluntary assistance to participate and each individual gave written informed consent before entry into the study. The study was approved by the Ethics Committee of the Saxonian medical chamber (EK-allg-37/10-1). All procedures utilized in this study are in agreement with the 1975 Declaration of Helsinki. 10 ml of sterile water was filled into sterile urine cups. The full water volume was gargled for 10 seconds before spitting it back into the cup. Analyzed anonymized gargle lavages from hospitalized COVID-19 patients and round robin samples (INSTAND) were kept frozen below -80°C for 1-2 months.

In solution capture purification of SARS-CoV-2 nucleic acids Per reaction 20µl Dynabeads MyOne Streptavidin C1 bead suspension (Thermo Fisher Scientific, #650.02) are pelleted using a magnetic rack and washed twice with 500µl combined 1x lysis/binding buffer (LysBB: 100 mM Tris-HCl, pH 7.5, 500 mM LiCl, 0.5% LiDS, 1 mM EDTA, 5 mM DTT). Washed beads are then resuspended in 100µl 1x LysBB before addition of 20pmol CV2_btn and 20pmol CV16_btn (see Supp. Table 1 

All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 6, 2020 . . https://doi.org/10.1101 for the RT-qPCR assay) and rotation for 10min at room temperature. Beads are pelleted and supernatant is discarded before resuspension in 200µl alkaline wash solution (125mM NaOH, and 5min incubation at room temperature. Beads are pelleted with a magnetic rack and supernatant is discarded. Beads are then washed once with 200µl 1x LysBB before resuspension in 500µl 2x LysBB. Prepared bead suspension can be stored at 5°C or used directly.

To 500µl ready-made bead suspension an equal volume of patient gargle lavage (sample), water (negative control) or sample with 500k copies of artificial SARS-CoV-2 RNA (Twist Biosciences, #102024) (positive and inhibition control) is added and mixed by pipetting. The reaction is incubated at 55°C for 10min and then placed on a magnetic rack. The supernatant is discarded and the beads are thoroughly resuspended in 200µl wash buffer (20 mM Tris-HCl, pH 7.5, 500 mM LiCl, 1 mM EDTA). Beads are pelleted and supernatant is discarded, repeat this step once for individual or inhibited samples. Wash beads once with 200µl low salt buffer (20 mM Tris-HCl, pH 7.5, 200 mM LiCl, 1 mM EDTA) and discard supernatant. Beads are resuspended in 25µl elution buffer (20 mM Tris-HCl, pH 7.5, 1 mM EDTA) and RNA is eluted by heating to 60°C for 2min. Transfer the supernatant to a fresh 0.2ml strip tube or use directly as input for RT-iLAMP or RT-qPCR.

For each sample two assays targeting either the Orf1a or N gene are performed. Primer mixes for both assays are prepared in bulk for quick reaction assembly (Supp Table 3 ). 20µl of RT-iLAMP master mix containing either the Orf1a (CV1-6) or N gene (CV15-20) primer sets (Supp.

Tables 4) and 10µl of capture eluate are combinded to obtain a final reaction volume of 30µl (1x WarmStart® Colorimetric LAMP Master Mix (NEB, Ipswich, MA, USA, #M1800L), 1mM ATP, 1µM SYTO 9, 1x primer mix, 0.1ng/µl Tte UvrD Helicase (NEB, #M1202S), 0.05U/µl thermostable inorganic pyrophosphatase (NEB, #M0296L), 0.4U/µl Protector RNase inhibitor (Sigma Aldrich, St. Louis, MI, USA, # 03335402001)). For color reactions, 0.5µl SYBR green I is applied to the lid of the tube without getting into contact with the reaction liquid. For transport or preparation of large numbers of reactions it is also possible to immobilize the dye by drying for 15min at 70°C in an oven. If amplification curves should be recorded on a qPCR machine, no SYBR green I is needed. The reaction is incubated at 65°C for 40min. If SYBR green I was applied to the cap of the tube, the reaction is then stopped by shaking and can be evaluated by eye or with any 'camera color-picker' app on a smartphone within a minute. We used the 'Palette Cam' app to score RGB values and extracted the hue. This can be easily done using a web tool (e.g. https://www.rapidtables.com/convert/color/rgb-to-hsv.html). Hue can also be scored directly when using e.g. 'Color Grab (color detection)' app for android or 'Aurora' app for iOS. All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 6, 2020 . . https://doi.org/10.1101 (D) Capture efficiency was estimated relative to automated silica-based RNA extraction based on copy number estimates for RT-qPCR assay targeting the SARS-CoV-2 E gene. (E) Equipment necessary for Cap-iLAMP: Pipettes and pipette tips, pre-mixed reagents (including iLAMP master mix and capture bead suspension), stable buffers (lysis/binding buffer,wash buffer, low salt buffer, elution buffer), a magnetic rack and a thermoblock.

All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 6, 2020 . . https://doi.org/10.1101 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 6, 2020. . https://doi.org/10.1101/2020.08.04.20168617 doi: medRxiv preprint Table 1 : Comparison of SARS-CoV-2 detection methods. Ten important aspects of different published SARS-CoV-2 detection methods and Cap-iLAMP are compared and ranked optimal (green), sub-optimal (light green), and critical (orange).

All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 6, 2020. . https://doi.org/10.1101/2020.08.04.20168617 doi: medRxiv preprint Supplementary Table 2 : Evaluation of Cap-iLAMP results. For each of the two assays (targeting either the SARS-CoV-2 Orf1a or N gene), a water negative control as well as a sample-specific positive/inhibition control comprised of sample and artificial viral RNA should be included. 

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The copyright holder for this this version posted August 6, 2020. . https://doi.org/10.1101/2020.08.04.20168617 doi: medRxiv preprint Supplementary Fig. 3 . Evaluation of different amounts of Tte UvrD helicase in LAMP reactions. Amplification plots of negative controls (left panel) and positive controls containing 1000 copies of artificial SARS-CoV-2 RNA (right panel). Amount of Tte UvrD helicase in 20µl reaction are indicated on the right. The primer set used targeted the N gene (CV15-20), the assay proceeded at 65°C. Fig. 4 Direct LAMP of patient gargle lavage samples can lead to false positives and false negatives. (A) Hues measured for negative (n=6) and positive (n=6) controls after RT-iLAMP reactions (left side) and after RT-iLAMP of SARS-CoV-2-negative (n=148) gargle lavage samples directly added to the reaction mix. (B) Hues measured after RT-iLAMP reactions preceeded by a λ exonuclease (NEB) digestion step (5U per reaction for 10-15min at 25°C) for negative (n=6) and positive (n=6) controls (left side), SARS-CoV-2-negative (n=100) and SARS-CoV-2-positive (n=7) gargle lavage samples directly added to the reaction mix (middle) and SARS-CoV-2negative (n=1) and SARS-CoV-2-positive (n=7) gargle lavage samples directly added to the reaction mix digested with QuickExtract (QE) and heat inactivated for 5min at 95°C before LAMP (right). Schematic drawing of λ exonuclease digestion shown on the right. Primers and LAMP reaction products do not possess a 5'-phosphate and are thus not a target for λ exonuclease digestion. All reactions contained 10µg T4 gene 32 protein (NEB) as it could alleviate inhibition of some gargle lavages in initial experiments. All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 6, 2020. . https://doi.org/10.1101/2020.08.04.20168617 doi: medRxiv preprint

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Supplementary Fig. 1. Sensitivity of different primer sets targeting SARS-CoV-2 RNA. (A) Amplification curves

We thank the management of Diakonische Dienste Leipzig, the personnel and residents of Altenpflegeheim Emmaus in Leipzig, and the personnel of the Department of Laboratory Medicine at Hospital St Georg Leipzig for their cooperation. Funding was provided by the Max Planck Society, the NOMIS foundation, and the ImmunoDeficiencyCenter Leipzig.

The authors declare no conflict of interest.