key: cord-1027362-jttp82uo
authors: Badbaran, Anita; Mailer, Reiner; Dahlke, Christine; Woens, Jannis; Fathi, Anahita; Mellinghoff, Sibylle C.; Renné, Thomas; Addo, Marylyn M.; Riecken, Kristoffer; Fehse, Boris
title: Digital PCR to quantify ChAdOx1 nCoV-19 copies in blood and tissues
date: 2021-05-28
journal: bioRxiv
DOI: 10.1101/2021.05.28.446155
sha: 5deb2a1b7dee7a20588d8c212723cfeb125eb3dd
doc_id: 1027362
cord_uid: jttp82uo

Vaccination with the adenoviral-vector based Astra Zeneca ChAdOx1 nCov-19 vaccine is efficient and safe. However, in rare cases vaccinated individuals developed life-threatening thrombotic complications, including thrombosis in cerebral sinus and splanchnic veins. Monitoring of the applied vector in vivo represents an important precondition to study the molecular mechanisms underlying vaccine-driven adverse effects now referred to as vaccine-induced immune thrombotic thrombocytopenia (VITT). We previously have shown that digital PCR is an excellent tool to quantify transgene copies in vivo. Here we present a highly sensitive digital PCR for in-situ quantification of ChAdOx1 nCoV-19 copies. Using this method, we quantified vector copies in human serum 24, 72 and 168 hours post vaccination, and in a variety of murine tissues in an experimental vaccination model 30 minutes post injection. We describe a method for high-sensitivity quantitative detection of ChAdOx1 nCoV-19 with possible implications to elucidate the mechanisms of severe ChAdOx1 nCov-19 vaccine complications.

Vaccination has been shown to be effective against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). 1 Adenoviral-vector based vaccines represent one of the cornerstones of the ongoing vaccination programs worldwide. Unfortunately, in contrast to their good safety profile, single cases of severe thromboembolism, often combined with thrombocytopenia were observed for the adenoviral-vector based vaccines from Astra Zeneca (ChAdOx1 nCov-19) [2] [3] [4] and, even more rarely, Johnson and Johnson (Ad26.COV2.S). 5 By beginning of April 2021, EudraVigilance reported a total of 169 cases of cerebral venous sinus thrombosis (CVST) and 53 cases of splanchnic vein thrombosis after vaccination with ChAdOx1 nCov-19. 6 This severe complication now referred to as vaccine-induced immune thrombotic thrombocytopenia (VITT, synonym TTS) to some extent resembles atypical heparin-induced thrombocytopenia (HIT) involving platelet-activating antibodies against platelet factor (PF) 4. 3, 4 Vector spread to distinct tissues might contribute to acute, but potentially also long-term side effects, e.g. caused by spontaneous insertion events. 7 Therefore, it is critical to develop methods that facilitate sensitive analysis of ChAdOx1 nCov-19 vector distribution. A better understanding might help to develop screening approaches for the progression of severe and serious adverse events and thus support prevention and early intervention measures.

We reasoned that digital PCR 8 should be well suited to detect and quantify vector copies after vaccination in humans, but also in experimental animal models. We previously showed applicability of dPCR to the detection of gene-modified cells in humans. 9

To decipher a part of the primary DNA sequence of the Spike coding sequence we designed suitable primers for nested PCR based on the known amino-acid sequence. 200 ng of genomic DNA isolated from blood cells drawn 45 min after vaccination with ChAdOx1 nCov-19 were used as template for the first reaction, 5 µl of a 1:50 dilution for the second. Both first and nested PCRs were performed in 100 µl final volume using Platinum PCR Supermix (ThermoFisher, Kandel, Germany) following a 40-cycle protocol with the following conditions: Initial denaturation: 94°C for 2 minutes; first 10 cycles: 94°C for 30 seconds, 50°C for 45 seconds, 72°C for 30 seconds; next 30 cycles: annealing temperature changed to 60°C; final extension -7 minutes. Both primary and nested PCR products were visualized on an agarose gel. A single band of the expected size (360 bp) was found after nested PCR and Sanger sequenced (Eurofins, Ebersberg, Germany). The DNA sequence located between the primers and the corresponding amino-acid sequence obtained by in-silico translation are depicted in Figure 1 . As evident, the amino-acid sequence showed 100% homology to the respective part of the published Spike sequence 10 ( Figure 1 ).

PCR primers and probes were designed with Primer Express version 3.0.1 (Thermo Fisher). All primers and probes were purchased from Eurofins. 

Digital PCRs were carried out as duplex reactions and analyzed with the QX100 Droplet Digital PCR System (Bio-Rad, California). 9, 11 For human samples, an assay detecting the human ribonuclease P/MRP subunit p30 RPP30 gene (Bio-Rad Assay ID dHsaCP2500350) was used as reference (probe: HEX-BHQ1); for murine cells we included our in-house established reference assay detecting the murine EpoR gene (based on 12 ) . In order to investigate gDNA amounts >60 ng, we added 5 U of the restriction enzymes MseI and ApaI (both Thermo Fisher) per individual reaction for human gDNA and mouse gDNA, respectively. PCR conditions followed the general Bio-Rad recommendations. 9 Data was analyzed with QuantaSoft_v1.7 software (Bio-Rad) including automatic Poisson correction. 9, 11 Digital-PCR testing

In order to assess sensitivity and specificity of the dPCR we used a dilution series of the PCR product of the first amplification round of the nested PCR; 16 different concentrations were tested. Those samples with expected copy numbers below 10 were tested in triplicates, and mean values were used to build the dilution curve. To test specificity, ten gDNA samples from healthy, non-vaccinated donors were tested in the duplex dPCR -all were highly positive for the REF gene, but negative for ChAdOx1 nCov-19.

Blood samples from nine volunteers were collected at the University Medical Center Hamburg-Eppendorf after their prime vaccination. Donors provided written informed consent (Ethical approval: PV4780). Volunteers received the vaccine ChAdOx1 nCov-19. At days 1, 3 and 7 post vaccination, EDTAblood was drawn and centrifuged at 200 x g for 10 min at RT. Afterwards, plasma was transferred and was again centrifuged at 1000 x g for 15 min at RT. Supernatant was stored at -80 °C.

C57Bl/6 mice of both sexes were anesthetized by intraperitoneal injection of ketamine (120 μg/g BW) and xylazine (16 μg/g BW in saline, 10 μl/g BW) and 50 μl ChAdOx1 nCov-19 vaccine were intradermally injected into the dorsal region. After 30 min mice were sacrificed by cervical dislocation and tissue samples were collected as described. 13 Citrated blood samples were centrifuged at 1000 x g for 10 min at 4°C, and gDNA was separately isolated from centrifuged cells (incl. thrombocytes) and plasma. Data is shown for blood cells. Mice were treated according to national guidelines for animal care at the animal facilities of University Medical Center Hamburg-Eppendorf and approved by local authorities (#56/18). All procedures were conducted in accordance with 3Rs regulations.

In order to identify an appropriate primer-probe combination we first deciphered parts of the primary DNA sequence of the ChAdOx1 nCov-19 vector. We designed a nested PCR located in a suitable region of the Spike-protein coding sequence 10 Figure 3a . Taken together, mean copy numbers were more than two times higher at 72 hours as compared to 24 hours post vaccination. In summary, this data proves the usefulness of the presented dPCR assay to monitor the presence of ChAdOx1 nCov-19 vector derived DNA in human blood after regular vaccination.

Furthermore, we evaluated whether dPCR might also be applied to detect ChAdOx1 nCov-19 vector copies in different tissues after experimental vaccination. We intradermally injected 50 µl of ChAdOx1 nCov-19 vaccine into the dorsal skin of C57Bl/6 mice. 13 Challenged animals were sacrificed 30 min later, organs were excised and genomic DNA was isolated from distinct organs/tissues as indicated in Figure   3 . Duplex dPCR was performed using the murine epoR gene as reference. As expected, huge numbers of ChAdOx1 nCov-19 vector copies were found at the injection site. Detection of ChAdOx1 nCov-19

vector copies in all tissues analyzed provided evidence for imminent vector spread into the blood stream and different organ tissues (Figure 3b ).

In summary, we have developed a dPCR assay that facilitates sensitive quantification of ChAdOx1 nCov-19 DNA copies. The assay was successfully applied to quantify ChAdOx1 nCov-19 copies in plasma of 

The authors declare no conflict of interest. ChAdOx1 nCoV-19 copies/ ml RPP 30 copies / ml

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We thank Hanna Thode for expert technical assistance and the laboratory team at the UKE, especially My Linh Ly, Monika Friedrich and Niclas Reneviér for collecting and processing the blood from volunteers as well as Amelie Sophie Alberti for study coordination. We acknowledge support by the