Determinants of protection against SARS-CoV-2 Omicron BA.1 and Delta infections in fully vaccinated outpatients (preprint)

We aimed to evaluate the association between the humoral and cellular immune responses and symptomatic SARS-CoV-2 infection with Delta or Omicron BA.1 variants in fully vaccinated outpatients. Anti-RBD IgG levels and IFN-{gamma} release were evaluated at PCR-diagnosis of SARS-CoV-2 in 636 samples from negative and positive patients during Delta and Omicron BA.1 periods. Median levels of anti-RBD IgG in positive patients were significantly lower than in negative patients for both variants (p < 0.05). The risk of Delta infection was inversely correlated with anti-RBD IgG titres (aOR = 0.63, 95% CI [0.41; 0.95], p = 0.03) and it was lower in the hybrid immunity group compared to the homologous vaccination group (aOR = 0.22, 95% CI [0.05; 0.62], p = 0.01). In contrast, neither the vaccination scheme nor anti-RBD IgG titers were associated with the risk of BA.1 infection in multivariable analysis. IFN-{gamma} release post-SARS-CoV-2 peptide stimulation was not different between samples from patients infected (either with Delta or Omicron BA.1 variant) or not (p = 0.77). Our results show that high circulating levels of anti-RBD IgG and hybrid immunity were independently associated with a lower risk of symptomatic SARS-CoV-2 infection in outpatients with differences according to the infecting variant.

Detection and prevalence of SARS-CoV-2 co-infections during the Omicron variant circulation, France, December 2021 - February 2022 (preprint)

In Dec 2021-Feb 2022, an intense and unprecedented co-circulation of SARS-CoV-2 variants with high genetic diversity raised the question of possible co-infections between variants and how to detect them. Using 11 mixes of Delta:Omicron isolates at different ratios, we evaluated the performance of 4 different sets of primers used for whole-genome sequencing and we developed an unbiased bioinformatics method which can detect all co-infections irrespective of the SARS-CoV-2 lineages involved. Applied on 21,387 samples collected between weeks 49-2021 and 08-2022 from random genomic surveillance in France, we detected 53 co-infections between different lineages. The prevalence of Delta and Omicron (BA.1) co-infections and Omicron lineages BA.1 and BA.2 co-infections were estimated at 0.18% and 0.26%, respectively. Among 6,242 hospitalized patients, the intensive care unit (ICU) admission rates were 1.64%, 4.81% and 15.38% in Omicron, Delta and Delta/Omicron patients, respectively. No BA.1/BA.2 co-infections were reported among ICU admitted patients. Although SARS-CoV-2 co-infections were rare in this study, their proper detection is crucial to evaluate their clinical impact and the risk of the emergence of potential recombinants.

Sars-Cov-2 Inactivation by a Panel of Commercialized Buffers and by Traditional Protocols (Heat or SDS) (preprint)

Following the rapid spread of COVID-19 across the globe, the intense response that was demanded of diagnostic centers and research laboratories prompted the use of numerous products and protocols for the management of SARS-CoV-2 specimens. In these settings, proper handling of such infectious specimen is necessary to ensure the safety of personnel and to reduce the risk of active transmission. Our aim was to evaluate the inactivation efficacy of different inactivating methods, notably from commercial lysis buffers available in diagnostic kits. Heat and sodium dodecyl sulfate detergent were also included in our investigations. A cell culture-based assay was used, and supported by molecular qRT-PCR detection, to show in vitro infectivity reduction after inactivation treatment. Overall, all the investigated methods were successful in inactivating SARS-CoV-2. Ten minutes of contact with the commercial buffers completely stopped in vitro SARS-CoV-2 infectivity. Fifteen minutes at 68°C and 30 minutes at 56°C as well as one hour with sodium dodecyl sulfate detergent at 2, 1, 0.5, and 0.1% yielded the same results. These findings demonstrate the reliability of these protocols with regards to biosafety. Inactivation by heat and sodium dodecyl sulfate detergent are rather simple and can be readily available methods for rendering an infectious SARS-CoV-2 specimen inactive, especially in settings where commercial buffers are not available.

Immunogenicity and efficacy of heterologous ChadOx1/BNT162b2 vaccination (preprint)

Following severe adverse reactions in patients vaccinated with the AstraZeneca ChadOx1 (Chad) vaccine, European health authorities have recommended that patients under the age of 55 who received one dose of Chad vaccine receive a second dose of Pfizer BNT162b2 (BNT) vaccine as a booster. However, the effectiveness and the immunogenicity of this vaccination regimen have not been formally tested. Here, we show that the heterologous Chad/BNT combination confers better protection against SARS-CoV-2 infection than the homologous BNT/BNT combination in a population of health care workers. To understand the underlying mechanism, we monitored in a longitudinal way the anti-spike immunity conferred by each vaccinal combination. Both combinations induced strong anti-spike antibody responses after boost in all vaccinated individuals. However, sera from heterologous vaccinated individuals displayed a stronger neutralizing activity, regardless of the SARS-CoV-2 variant analyzed, and this was associated with more switched memory RBD-specific B cells with an activated phenotype and less IgA. The Chad vaccine induced a stronger T cell response than the BNT vaccine after the priming dose, and the reciprocal was true for the IgG response, which could explain the complementarity of both vaccines when used in an heterologous setting. This strongly protective vaccination regimen could be therefore particularly suitable for immunocompromised individuals.

Immunity to SARS-CoV-2 in a Dialyzed Patient Who Developed COVID-19 Twenty Days After the Second Dose of BNT162b2 Vaccine: a Case Report (preprint)

Introduction: End stage kidney disease (ESKD) and cancer have been identified as risk factors for severe and fatal cases of COVID-19, making vaccination in these patients a priority. Patients suffering from ESKD have a significantly weaker response to common vaccines than general population. However, humoral and cellular immune responses after two doses of RNA-based vaccine BNT162b2 (Pfizer–BioNTech) have been poorly explored in this vulnerable population.Case presentationA 69-year-old male patient was followed for ESKD and myeloma. He developed a severe SARS-CoV-2 pneumonia twenty days after two doses of BNT162b2 vaccine. Whole genome sequencing found that the virus belonged to the 20I/501Y.V1 clade. A serology draws eight days after the 2 nd vaccine dose showed positive RBD IgG without neutralizing activity. A serum specimen sampled thirty days after the onset of SARS-CoV-2 infection showed seroconversion against both RBD and N antigens. This specimen was shown to exhibit a frank neutralizing activity. The QuantiFERON® SARS-CoV-2 (Qiagen) showed a positive specific cellular response although the QuantiFERON monitor displayed a weak cellular response. ConclusionsImpaired immunity due to renal failure probably explain the severe pneumonia despite vaccination. The fact that the patient developpe a neutralizing activity and a cellular response after a third stimulation by infection may suggest to systemically administrate a third dose of vaccine in ESKD patients.

Live virus neutralization testing in convalescent and vaccinated subjects against 19A, 20B, 20I/501Y.V1 and 20H/501.V2 isolates of SARS-CoV-2 (preprint)

Background: SARS-CoV-2 mutations appeared recently and can lead to conformational changes in the spike protein and probably induce modifications in antigenicity. In this study, we wanted to assess the neutralizing capacity of antibodies to prevent cell infection, using a live virus neutralisation test. Methods: Sera samples were collected from different populations: two-dose vaccinated COVID-19-naive healthcare workers (HCWs; Pfizer-BioNTech BNT161b2), 6-months post mild COVID-19 HCWs, and critical COVID-19 patients. We tested various clades such as 19A (initial one), 20B (B.1.1.241 lineage), 20I/501Y.V1 (B.1.1.7 lineage), and 20H/501Y.V2 (B.1.351 lineage). Results: No significant difference was observed between the 20B and 19A isolates for HCWs with mild COVID-19 and critical patients. However, a significant decrease in neutralisation ability was found for 20I/501Y.V1 in comparison with 19A isolate for critical patients and HCWs 6-months post infection. Concerning 20H/501Y.V2, all populations had a significant reduction in neutralising antibody titres in comparison with the 19A isolate. Interestingly, a significant difference in neutralisation capacity was observed for vaccinated HCWs between the two variants whereas it was not significant for the convalescent groups. Conclusion: Neutralisation capacity was slightly reduced for critical patients and HCWs 6-months post infection. No neutralisation escape could be feared concerning the two variants of concern in both populations. The reduced neutralising response observed towards the 20H/501Y.V2 in comparison with the 19A and 20I/501Y.V1 isolates in fully immunized subjects with the BNT162b2 vaccine is a striking finding of the study.

Screening of the H69 and V70 deletions in the SARS-CoV-2 spike protein with a RT-PCR diagnosis assay reveals low prevalence in Lyon, France (preprint)

We report the implementation of a two-step strategy for the identification of SARS-CoV-2 variants carrying the spike deletion H69-V70 ({Delta}H69/{Delta}V70). This spike deletion resulted in a S-gene target failure (SGTF) of a three-target RT-PCR assay (TaqPath kit). Whole genome sequencing performed on 37 samples with SGTF revealed several receptor-binding domain mutations co-occurring with {Delta}H69/{Delta}V70. More importantly, this strategy enabled the first detection of the variant of concern 202012/01 in France on December 21th 2020. Since September a SARS-CoV-2 spike (S) deletion H69-V70 ({Delta}H69/{Delta}V70) has attracted increasing attention. This deletion was detected in the cluster-5 variant identified both in minks and humans in Denmark. This cluster-5 variant carries a receptor binding domain (RBD) mutation Y453F and was associated with reduced susceptibility to neutralizing antibodies to sera from recovered COVID-19 patients [1-3]. The {Delta}H69/{Delta}V70 has also co-occurred with two other RBD mutations of increasing interest [4]: N439K that is currently spreading in Europe and might also have reduced susceptibility to SARS-CoV-2 antibodies [5]; and N501Y that is part of the SARS-CoV-2 variant of concern (VOC) 202012/01 recently detected in England [6]. Although the impact of {Delta}H69/{Delta}V70 on SARS-CoV-2 pathogenesis is not clear, enhanced surveillance is urgently needed. Herein we report the implementation of a two-step strategy enabling a rapid detection of VOC 202012/01 or other variants carrying {Delta}H69/{Delta}V70.

Characterization of SARS-CoV-2 ORF6 deletion variants detected in a nosocomial cluster during routine genomic surveillance, Lyon, France (preprint)

Through routine genomic surveillance of the novel SARS-CoV-2 virus (n=229 whole genome sequences), 2 different frameshifting deletions were newly detected in the open reading frame (ORF) 6, starting at the same position (27267). While the 26-nucleotide deletion variant was only found in one sample in March 2020, the 34-nucleotide deletion variant was found within a single geriatric hospital unit in 5/9 patients sequenced and one health care worker with samples collected between April 2nd and 9th, 2020. Both the presence of the 34-nucleotide deletion variant limited to this unit and the clustering of the corresponding whole genome sequences by phylogeny analysis strongly suggested a nosocomial transmission between patients. Interestingly, prolonged viral excretion of the 34-nucleotide deletion variant was identified in a stool sample 14 days after initial diagnosis for one patient. Clinical data revealed no significant difference in disease severity between patients harboring the wild-type or the 34-nucleotide deletion variants. The in vitro infection of the two deletion variants on primate endothelial kidney cells (BGM) and human lung adenocarcinoma cells (Calu-3) yielded comparable replication kinetics with the wild-type strain. Furthermore, high viral loads were found in vivo regardless of the presence or absence of the ORF6 deletion. Our study highlights the transmission and replication capacity of two newly described deletion variants in the same ORF6 region. ImportanceWhile the SARS-CoV-2 genome has remained relatively stable since its emergence in the human population, genomic deletions are an evolutionary pattern previously described for the related SARS-CoV. Real-time genomic monitoring of the circulating variants is paramount to detect strain prevalence and transmission dynamics. Given the role of ORF6 in interferon modulation, further characterization, such as mechanistic interactions and interferon monitoring in patients, is crucial in understanding the viral-host factors driving disease evolution.

Evaluation of NGS-based approaches for SARS-CoV-2 whole genome characterisation (preprint)

Since the beginning of the COVID-19 outbreak, SARS-CoV-2 whole-genome sequencing (WGS) has been performed at unprecedented rate worldwide with the use of very diverse Next Generation Sequencing (NGS) methods. Herein, we compare the performance of four NGS-based approaches for SARS-CoV-2 WGS. Twenty four clinical respiratory samples with a large scale of Ct values (from 10.7 to 33.9) were sequenced with four methods. Three used Illumina sequencing: an in-house metagenomic NGS (mNGS) protocol and two newly commercialized kits including a hybridization capture method developed by Illumina (DNA Prep with Enrichment kit and Respiratory Virus Oligo Panel, RVOP) and an amplicon sequencing method developed by Paragon Genomics (CleanPlex SARS-CoV-2 kit). We also evaluated the widely used amplicon sequencing protocol developed by ARTIC Network and combined with Oxford Nanopore Technologies (ONT) sequencing. All four methods yielded near-complete genomes (>99%) for high viral loads samples, with mNGS and RVOP producing the most complete genomes. For mid viral loads, 2/8 and 1/8 genomes were incomplete (<99%) with mNGS and both CleanPlex and RVOP, respectively. For low viral loads (Ct [≥]25), amplicon-based enrichment methods were the most sensitive techniques yielding complete genomes for 7/8 samples. All methods were highly concordant in terms of identity in complete consensus sequence. Just one mismatch in two samples was observed in CleanPlex vs the other methods, due to the dedicated bioinformatics pipeline setting a high threshold to call SNP compared to reference sequence. Importantly, all methods correctly identified a newly observed 34-nt deletion in ORF6 but required specific bioinformatic validation for RVOP. Finally, as a major warning for targeted techniques, a default of coverage in any given region of the genome should alert to a potential rearrangement or a SNP in primer annealing or probe-hybridizing regions and would require regular updates of the technique according to SARS-CoV-2 evolution.

Molecular characterization of SARS-CoV-2 in the first COVID-19 cluster in France reveals an amino-acid deletion in nsp2 (Asp268Del) (preprint)

We present the first genetic characterization of a COVID-19 cluster in Europe using metagenomic next-generation sequencing (mNGS). Despite low viral loads, the mNGS workflow used herein allowed to characterize the whole genome sequences of SARS-CoV2 isolated from an asymptomatic patient, in 2 clinical samples collected 1 day apart. Comparison of these sequences suggests viral evolution with development of quasispecies. In addition, the present workflow identified a new deletion in nsp2 (Asp268Del) which was found in all 3 samples originating from this cluster as well as in 37 other viruses collected in England and in Netherlands, suggesting the spread of this deletion in Europe. The impact of Asp268Del on SARS-CoV-2 transmission and pathogenicity, as well as on PCR performances and anti-viral strategy should be rapidly evaluated in further studies.