Maturation and persistence of the anti-SARS-CoV-2 memory B cell response.

Memory B cells play a fundamental role in host defenses against viruses, but to date, their role has been relatively unsettled in the context of SARS-CoV-2. We report here a longitudinal single-cell and repertoire profiling of the B cell response up to 6 months in mild and severe COVID-19 patients. Distinct SARS-CoV-2 spike-specific activated B cell clones fueled an early antibody-secreting cell burst as well as a durable synchronous germinal center response. While highly mutated memory B cells, including pre-existing cross-reactive seasonal Betacoronavirus-specific clones, were recruited early in the response, neutralizing SARS-CoV-2 RBD-specific clones accumulated with time and largely contributed to the late, remarkably stable, memory B cell pool. Highlighting germinal center maturation, these cells displayed clear accumulation of somatic mutations in their variable region genes over time. Overall, these findings demonstrate that an antigen-driven activation persisted and matured up to 6 months after SARS-CoV-2 infection and may provide long-term protection.

SARS-CoV-2 Omicron BA.1 breakthrough infection drives late remodeling of the memory B cell repertoire in vaccinated individuals.

How infection by a viral variant showing antigenic drift impacts a preformed mature human memory B cell (MBC) repertoire remains an open question. Here, we studied the MBC response up to 6 months after SARS-CoV-2 Omicron BA.1 breakthrough infection in individuals previously vaccinated with three doses of the COVID-19 mRNA vaccine. Longitudinal analysis, using single-cell multi-omics and functional analysis of monoclonal antibodies from RBD-specific MBCs, revealed that a BA.1 breakthrough infection mostly recruited pre-existing cross-reactive MBCs with limited de novo response against BA.1-restricted epitopes. Reorganization of clonal hierarchy and new rounds of germinal center reactions, however, combined to maintain diversity and induce progressive maturation of the MBC repertoire against common Hu-1 and BA.1, but not BA.5-restricted, SARS-CoV-2 Spike RBD epitopes. Such remodeling was further associated with a marked improvement in overall neutralizing breadth and potency. These findings have fundamental implications for the design of future vaccination booster strategies.

Analysis of mRNA vaccination-elicited RBD-specific memory B cells reveals strong but incomplete immune escape of the SARS-CoV-2 Omicron variant.

The SARS-CoV-2 Omicron variant can escape neutralization by vaccine-elicited and convalescent antibodies. Memory B cells (MBCs) represent another layer of protection against SARS-CoV-2, as they persist after infection and vaccination and improve their affinity. Whether MBCs elicited by mRNA vaccines can recognize the Omicron variant remains unclear. We assessed the affinity and neutralization potency against the Omicron variant of several hundred naturally expressed MBC-derived monoclonal IgG antibodies from vaccinated COVID-19-recovered and -naive individuals. Compared with other variants of concern, Omicron evaded recognition by a larger proportion of MBC-derived antibodies, with only 30% retaining high affinity against the Omicron RBD, and the reduction in neutralization potency was even more pronounced. Nonetheless, neutralizing MBC clones could be found in all the analyzed individuals. Therefore, despite the strong immune escape potential of the Omicron variant, these results suggest that the MBC repertoire generated by mRNA vaccines still provides some protection against the Omicron variant in vaccinated individuals.

mRNA vaccination of naive and COVID-19-recovered individuals elicits potent memory B cells that recognize SARS-CoV-2 variants.

In addition to serum immunoglobulins, memory B cell (MBC) generation against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is another layer of immune protection, but the quality of MBC responses in naive and coronavirus disease 2019 (COVID-19)-recovered individuals after vaccination remains ill defined. We studied longitudinal cohorts of naive and disease-recovered individuals for up to 2 months after SARS-CoV-2 mRNA vaccination. We assessed the quality of the memory response by analysis of antibody repertoires, affinity, and neutralization against variants of concern (VOCs) using unbiased cultures of 2,452 MBCs. Upon boosting, the MBC pool of recovered individuals expanded selectively, matured further, and harbored potent neutralizers against VOCs. Although naive individuals had weaker neutralizing serum responses, half of their RBD-specific MBCs displayed high affinity toward multiple VOCs, including delta (B.1.617.2), and one-third retained neutralizing potency against beta (B.1.351). Our data suggest that an additional challenge in naive vaccinees could recall such affinity-matured MBCs and allow them to respond efficiently to VOCs.

High fusion and cytopathy of SARS-CoV-2 variant B.1.640.1.

SARS-CoV-2 variants with undetermined properties have emerged intermittently throughout the COVID-19 pandemic. Some variants possess unique phenotypes and mutations which allow further characterization of viral evolution and Spike functions. Around 1,100 cases of the B.1.640.1 variant were reported in Africa and Europe between 2021 and 2022, before the expansion of Omicron. Here, we analyzed the biological properties of a B.1.640.1 isolate and its Spike. Compared to the ancestral Spike, B.1.640.1 carried 14 amino acid substitutions and deletions. B.1.640.1 escaped binding by some anti-N-terminal domain and anti-receptor-binding domain monoclonal antibodies, and neutralization by sera from convalescent and vaccinated individuals. In cell lines, infection generated large syncytia and a high cytopathic effect. In primary airway cells, B.1.640.1 replicated less than Omicron BA.1 and triggered more syncytia and cell death than other variants. The B.1.640.1 Spike was highly fusogenic when expressed alone. This was mediated by two poorly characterized and infrequent mutations located in the Spike S2 domain, T859N and D936H. Altogether, our results highlight the cytopathy of a hyper-fusogenic SARS-CoV-2 variant, supplanted upon the emergence of Omicron BA.1. (This study has been registered at ClinicalTrials.gov under registration no. NCT04750720.)IMPORTANCEOur results highlight the plasticity of SARS-CoV-2 Spike to generate highly fusogenic and cytopathic strains with the causative mutations being uncharacterized in previous variants. We describe mechanisms regulating the formation of syncytia and the subsequent consequences in a primary culture model, which are poorly understood.

Clinical Phenotypes and Molecular Characteristics of Respiratory Syncytial Virus in Adults: A Monocentric Prospective Study Between 2019 and 2022.

Respiratory syncytial virus (RSV) infection is a major cause of pneumonia in adults. Little is known on the viral genetic diversity and the associated clinical phenotypes in this population. This single-center prospective cohort study included RSV-infected patients hospitalized between January 2019 and December 2022. Of 100 patients, including 41 with severe infection, 72 were infected with RSV-B. RSV genome sequencing showed no clustering according to severity. Patients infected with RSV-B with risk factors for severe pneumonia had significantly higher fusion protein diversity scores. No amino acid substitutions conferring resistance to nirsevimab were detected.

'Unusual' HCV genotype subtypes: origin, distribution, sensitivity to direct-acting antiviral drugs and behaviour on antiviral treatment and retreatment.

The high genetic diversity of hepatitis C virus (HCV) has led to the emergence of eight genotypes and a large number of subtypes in limited geographical areas. Currently approved pangenotypic DAA regimens have been designed and developed to be effective against the most common subtypes (1a, 1b, 2a, 2b, 2c, 3a, 4a, 5a and 6a). However, large populations living in Africa and Asia, or who have migrated from these regions to industrialised countries, are infected with 'unusual', non-epidemic HCV subtypes, including some that are inherently resistant to currently available direct-acting antiviral (DAA) drugs due to the presence of natural polymorphisms at resistance-associated substitution positions. In this review article, we describe the origin and subsequent global spread of HCV genotypes and subtypes, the current global distribution of common and unusual HCV subtypes, the polymorphisms naturally present in the genome sequences of unusual HCV subtypes that may confer inherently reduced susceptibility to DAA drugs and the available data on the response of unusual HCV subtypes to first-line HCV therapy and retreatment. We conclude that the problem of unusual HCV subtypes that are inherently resistant to DAAs and its threat to the global efforts to eliminate viral hepatitis are largely underestimated and warrant vigorous action.

Virological characterization of treatment failures and retreatment outcomes in patients infected with "unusual" HCV genotype 1 subtypes.

BACKGROUND AND AIMS: Suboptimal rates of sustained virological response have been reported in patients infected with an "unusual," non-1a/1b HCV genotype 1 subtype. The objectives of this study were to assess the proportion of non-1a/1b genotype 1 subtypes in a population of HCV-infected patients who failed to achieve sustained virological response after first-line direct-acting antiviral treatment, to virologically characterize their failures and to assess their outcomes on retreatment. APPROACH AND RESULTS: Samples addressed between January 2015 and December 2021 to the French National Reference Center for Viral Hepatitis B, C, and D were prospectively analyzed by means of Sanger and deep sequencing. Among 640 failures, 47 (7.3%) occurred in patients infected with an "unusual" genotype 1 subtype. Samples were available in 43 of them; 92.5% of these patients were born in Africa. Our results show the presence at baseline and at treatment failure of NS3 protease and/or NS5A polymorphisms conferring inherent reduced susceptibility to direct-acting antivirals in these patients, together with the presence at failure of additional resistance-associated substitutions not naturally present as dominant species, but jointly selected by first-line therapy. CONCLUSIONS: Patients infected with "unusual" HCV genotype 1 subtypes are over-represented among direct-acting antiviral treatment failures. Most of them were born and likely infected in sub-Saharan Africa. "Unusual" HCV genotype 1 subtypes naturally carry polymorphisms that confer reduced susceptibility to the drugs currently used to cure hepatitis C, in particular the NS5A inhibitors. Retreatment with sofosbuvir plus an NS3 protease and an NS5A inhibitor is generally efficacious.

Regional dissemination of NDM-1 producing Enterobacter hormaechei ST1740, with a subset of strains co-producing VIM-4 or IMP-13, France, 2019 to 2022.

BackgroundFrom 2019 to 2022, the French National Reference Centre for Antibiotic Resistance (NRC) received a total of 25 isolates of Enterobacter hormaechei subsp. hoffmannii sequence type (ST)1740. All produced metallo-ß-lactamase(s) and were from the Lyon area.AimTo understand these strains' spread and evolution, more extended microbiological and molecular analyses were conducted.MethodsPatients' demographics and specimen type related to isolates were retrieved. All strains underwent short-read whole genome sequencing, and for 15, long-read sequencing to understand carbapenemase-gene acquisition. Clonal relationships were inferred from core-genome single nt polymorphisms (SNPs). Plasmids and the close genetic environment of each carbapenemase-encoding gene were analysed.ResultsPatients (10 female/15 male) were on average 56.6 years old. Seven isolates were recovered from infections and 18 through screening. With ≤ 27 SNPs difference between each other's genome sequences, the 25 strains represented a clone dissemination. All possessed a chromosome-encoded bla NDM-1 gene inside a composite transposon flanked by two IS3000. While spreading, the clone independently acquired a bla VIM-4-carrying plasmid of IncHI2 type (n = 12 isolates), or a bla IMP-13-carrying plasmid of IncP-1 type (n = 1 isolate). Of the 12 isolates co-producing NDM-1 and VIM-4, seven harboured the colistin resistance gene mcr9.2; the remaining five likely lost this gene through excision.ConclusionThis long-term outbreak was caused by a chromosome-encoded NDM-1-producing ST1740 E. hormaechei subsp. hoffmannii clone, which, during its dissemination, acquired plasmids encoding VIM-4 or IMP-13 metallo-ß-lactamases. To our knowledge, IMP-13 has not prior been reported in Enterobacterales in France. Epidemiological and environmental investigations should be considered alongside microbiological and molecular ones.

Unknown Circovirus in Immunosuppressed Patient with Hepatitis, France, 2022.

Hepatitis of undetermined origin can be caused by a wide variety of pathogens, sometimes emerging pathogens. We report the discovery, by means of routine shotgun metagenomics, of a new virus belonging to the family Circoviridae, genus Circovirus, in a patient in France who had acute hepatitis of unknown origin.

Viral genomic, metagenomic and human transcriptomic characterization and prediction of the clinical forms of COVID-19.

COVID-19 is characterized by respiratory symptoms of various severities, ranging from mild upper respiratory signs to acute respiratory failure/acute respiratory distress syndrome associated with a high mortality rate. However, the pathophysiology of the disease is largely unknown. Shotgun metagenomics from nasopharyngeal swabs were used to characterize the genomic, metagenomic and transcriptomic features of patients from the first pandemic wave with various forms of COVID-19, including outpatients, patients hospitalized not requiring intensive care, and patients in the intensive care unit, to identify viral and/or host factors associated with the most severe forms of the disease. Neither the genetic characteristics of SARS-CoV-2, nor the detection of bacteria, viruses, fungi or parasites were associated with the severity of pulmonary disease. Severe pneumonia was associated with overexpression of cytokine transcripts activating the CXCR2 pathway, whereas patients with benign disease presented with a T helper "Th1-Th17" profile. The latter profile was associated with female gender and a lower mortality rate. Our findings indicate that the most severe cases of COVID-19 are characterized by the presence of overactive immune cells resulting in neutrophil pulmonary infiltration which, in turn, could enhance the inflammatory response and prolong tissue damage. These findings make CXCR2 antagonists, in particular IL-8 antagonists, promising candidates for the treatment of patients with severe COVID-19.

Omicron induced distinct immune respiratory transcriptomics signatures compared to pre-existing variants in critically ill COVID-19 patients.

Severe coronavirus disease 2019 (COVID-19) is related to dysregulated immune responses. We aimed to explore the effect of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants on the immune response by nasopharyngeal transcriptomic in critically-ill patients. This prospective monocentric study included COVID-19 patients requiring intensive care unit (ICU) admission between March 2020 and 2022. Patients were classified according to VOC (ancestral, Alpha, Delta, and Omicron). Eighty-eight patients with severe COVID-19 were included after matching (on prespecified clinical criteria). Profiling of gene expression markers of innate and adaptive immune responses were investigated by respiratory transcriptomics at ICU admission. Eighty-eight patients were included in the study after matching (ancestral [n = 24], Alpha [n = 24], Delta [n = 22], and Omicron [n = 18] variants). Respiratory transcriptomic analysis revealed distinct innate and adaptive immune profiling between variants. In comparison with the ancestral variant, there was a reduced expression of neutrophil degranulation, T cell activation, cytokines signalling pathways in patients infected with Alpha and Delta variants. In contrast, there was a higher expression of neutrophil degranulation, T and B cells activation, and inflammatory interleukins pathways in patients infected with Omicron. To conclude, Omicron induced distinct immune respiratory transcriptomics signatures compared to pre-existing variants in patients with severe COVID-19, pointing to an evolving pathophysiology of severe COVID-19 in the Omicron era.

Emergence and rapid dissemination of highly resistant NDM-14-producing Klebsiella pneumoniae ST147, France, 2022.

BackgroundSince 2021, an emergence of New Delhi metallo-ß-lactamase (NDM)-14-producing Klebsiella pneumoniae has been identified in France. This variant with increased carbapenemase activity was not previously detected in Enterobacterales.AimWe investigated the rapid dissemination of NDM-14 producers among patients in hospitals in France.MethodsAll NDM-14-producing non-duplicate clinical isolates identified in France until June 2022 (n = 37) were analysed by whole genome sequencing. The phylogeny of NDM-14-producers among all K. pneumoniae sequence type (ST) 147 reported in France since 2014 (n = 431) was performed. Antimicrobial susceptibility testing, conjugation experiments, clonal relationship and molecular clock analysis were performed.ResultsThe 37 NDM-14 producers recovered in France until 2022 belonged to K. pneumoniae ST147. The dissemination of NDM-14-producing K. pneumoniae was linked to a single clone, likely imported from Morocco and responsible for several outbreaks in France. The gene bla NDM-14 was harboured on a 54 kilobase non-conjugative IncFIB plasmid that shared high homology with a known bla NDM-1-carrying plasmid. Using Bayesian analysis, we estimated that the NDM-14-producing K. pneumoniae ST147 clone appeared in 2020. The evolutionary rate of this clone was estimated to 5.61 single nucleotide polymorphisms per genome per year. The NDM-14 producers were highly resistant to all antimicrobials tested except to colistin, cefiderocol (minimum inhibitory concentration 2 mg/L) and the combination of aztreonam/avibactam.ConclusionHighly resistant NDM-14 producing K. pneumoniae can rapidly spread in healthcare settings. Surveillance and thorough investigations of hospital outbreaks are critical to evaluate and limit the dissemination of this clone.

Small-Molecule Cyclophilin Inhibitors Potently Reduce Platelet Procoagulant Activity.

Procoagulant platelets are associated with an increased risk for thrombosis. Procoagulant platelet formation is mediated via Cyclophilin D (CypD) mediated opening of the mitochondrial permeability transition pore. Inhibiting CypD activity could therefore be an interesting approach to limiting thrombosis. In this study, we investigated the potential of two novel, non-immunosuppressive, non-peptidic small-molecule cyclophilin inhibitors (SMCypIs) to limit thrombosis in vitro, in comparison with the cyclophilin inhibitor and immunosuppressant Cyclosporin A (CsA). Both cyclophilin inhibitors significantly decreased procoagulant platelet formation upon dual-agonist stimulation, shown by a decreased phosphatidylserine (PS) exposure, as well as a reduction in the loss of mitochondrial membrane potential. Furthermore, the SMCypIs potently reduced procoagulant platelet-dependent clotting time, as well as fibrin formation under flow, comparable to CsA. No effect was observed on agonist-induced platelet activation measured by P-selectin expression, as well as CypA-mediated integrin αIIbß3 activation. Importantly, whereas CsA increased Adenosine 5'-diphosphate (ADP)-induced platelet aggregation, this was unaffected in the presence of the SMCypIs. We here demonstrate specific cyclophilin inhibition does not affect normal platelet function, while a clear reduction in procoagulant platelets is observed. Reducing platelet procoagulant activity by inhibiting cyclophilins with SMCypIs forms a promising strategy to limit thrombosis.

Apalutamide Prevents SARS-CoV-2 Infection in Lung Epithelial Cells and in Human Nasal Epithelial Cells.

In early 2020, the novel pathogenic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, China, and rapidly propagated worldwide causing a global health emergency. SARS-CoV-2 binds to the angiotensin-converting enzyme 2 (ACE2) protein for cell entry, followed by proteolytic cleavage of the Spike (S) protein by the transmembrane serine protease 2 (TMPRSS2), allowing fusion of the viral and cellular membranes. Interestingly, TMPRSS2 is a key regulator in prostate cancer (PCa) progression which is regulated by androgen receptor (AR) signaling. Our hypothesis is that the AR signaling may regulate the expression of TMPRSS2 in human respiratory cells and thus influence the membrane fusion entry pathway of SARS-CoV-2. We show here that TMPRSS2 and AR are expressed in Calu-3 lung cells. In this cell line, TMPRSS2 expression is regulated by androgens. Finally, pre-treatment with anti-androgen drugs such as apalutamide significantly reduced SARS-CoV-2 entry and infection in Calu-3 lung cells but also in primary human nasal epithelial cells. Altogether, these data provide strong evidence to support the use of apalutamide as a treatment option for the PCa population vulnerable to severe COVID-19.

Diagnostic Accuracy of Assays Using Point-of-Care Testing or Dried Blood Spot Samples for the Determination of Hepatitis C Virus RNA: A Systematic Review.

BACKGROUND: Finger-stick point-of-care and dried blood spot (DBS) hepatitis C virus (HCV) RNA testing increases testing uptake and linkage to care. This systematic review evaluated the diagnostic accuracy of point-of-care testing and DBS to detect HCV RNA. METHODS: Bibliographic databases and conference presentations were searched for eligible studies. Meta-analysis was used to pool estimates. RESULTS: Of 359 articles identified, 43 studies were eligible and included. When comparing the Xpert HCV Viral Load Fingerstick assay to venous blood samples (7 studies with 987 samples), the sensitivity and specificity for HCV RNA detection was 99% (95% confidence interval [CI], 97%-99%) and 99% (95% CI, 94%-100%) and for HCV RNA quantification was 100% (95% CI, 93%-100%) and 100% (95% CI, 94%-100%). The proportion of invalid results following Xpert HCV Viral Load Fingerstick testing was 6% (95% CI, 3%-11%). When comparing DBS to venous blood samples (28 studies with 3988 samples) the sensitivity and specificity for HCV RNA detection was 97% (95% CI, 95%-98%) and 100% (95% CI, 98%-100%) and for HCV RNA quantification was 98% (95% CI, 96%-99%) and 100% (95% CI, 95%-100%). CONCLUSIONS: Excellent diagnostic accuracy was observed across assays for detection of HCV RNA from finger-stick and DBS samples. The proportion of invalid results following Xpert HCV Viral Load Fingerstick testing highlights the importance of operator training and quality assurance programs.

BNT162b2 Messenger RNA Vaccination Did Not Prevent an Outbreak of Severe Acute Respiratory Syndrome Coronavirus 2 Variant 501Y.V2 in an Elderly Nursing Home but Reduced Transmission and Disease Severity.

We report an outbreak of severe acute respiratory syndrome coronavirus 2 501Y.V2 in a nursing home. All nonvaccinated residents (5/5) versus half of those vaccinated with BNT162b2 (13/26) were infected. Two of 13 vaccinated versus 4 of 5 nonvaccinated residents presented severe disease. BNT162b2 did not prevent the outbreak, but reduced transmission and disease severity.

Neutralization Heterogeneity of UK and South African Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Variants in BNT162b2-Vaccinated or Convalescent Coronavirus Disease 2019 (COVID-19) Healthcare Workers.

There are concerns about neutralizing antibodies' (NAbs') potency against severe acute respiratory syndrome coronavirus 2 variants. Despite decreased NAb titers elicited by BNT162b2 vaccine against VOC202012/01 and 501Y.V2 strains, 28/29 healthcare workers (HCWs) had an NAb titer ≥1:10. In contrast, 6 months after coronavirus disease 2019 mild forms, only 9/15 (60%) of HCWs displayed detectable NAbs against 501Y.V2 strain.

Persistent SARS-CoV-2 Alpha Variant Infection in Immunosuppressed Patient, France, February 2022.

We describe persistent circulation of SARS-CoV-2 Alpha variant in an immunosuppressed patient in France during February 2022. The virus had a new pattern of mutation accumulation. The ongoing circulation of previous variants of concern could lead to reemergence of variants with the potential to propagate future waves of infection.

Artificial intelligence predicts immune and inflammatory gene signatures directly from hepatocellular carcinoma histology.

BACKGROUND & AIMS: Patients with hepatocellular carcinoma (HCC) displaying overexpression of immune gene signatures are likely to be more sensitive to immunotherapy, however, the use of such signatures in clinical settings remains challenging. We thus aimed, using artificial intelligence (AI) on whole-slide digital histological images, to develop models able to predict the activation of 6 immune gene signatures. METHODS: AI models were trained and validated in 2 different series of patients with HCC treated by surgical resection. Gene expression was investigated using RNA sequencing or NanoString technology. Three deep learning approaches were investigated: patch-based, classic MIL and CLAM. Pathological reviewing of the most predictive tissue areas was performed for all gene signatures. RESULTS: The CLAM model showed the best overall performance in the discovery series. Its best-fold areas under the receiver operating characteristic curves (AUCs) for the prediction of tumors with upregulation of the immune gene signatures ranged from 0.78 to 0.91. The different models generalized well in the validation dataset with AUCs ranging from 0.81 to 0.92. Pathological analysis of highly predictive tissue areas showed enrichment in lymphocytes, plasma cells, and neutrophils. CONCLUSION: We have developed and validated AI-based pathology models able to predict the activation of several immune and inflammatory gene signatures. Our approach also provides insights into the morphological features that impact the model predictions. This proof-of-concept study shows that AI-based pathology could represent a novel type of biomarker that will ease the translation of our biological knowledge of HCC into clinical practice. LAY SUMMARY: Immune and inflammatory gene signatures may be associated with increased sensitivity to immunotherapy in patients with advanced hepatocellular carcinoma. In the present study, the use of artificial intelligence-based pathology enabled us to predict the activation of these signatures directly from histology.