COVID-19 associated pulmonary aspergillosis in critically-ill patients: a prospective multicenter study in the era of Delta and Omicron variants.

BACKGROUND: During the first COVID-19 pandemic wave, COVID-19-associated pulmonary aspergillosis (CAPA) has been reported in up to 11-28% of critically ill COVID-19 patients and associated with increased mortality. As new SARS-CoV-2 variants emerged, the characteristics of critically ill COVID-19 patients have evolved, particularly in the era of Omicron. The purpose of this study is to investigate the characteristics of CAPA in the era of new variants. METHODS: This is a prospective multicenter observational cohort study conducted in France in 36 participating intensive care units (ICU), between December 7th, 2021 and April 26th 2023. Diagnosis criteria of CAPA relied on European Confederation of Medical Mycology (ECMM)/International Society for Human & Animal Mycology (ISHAM) consensus criteria. RESULTS: 566 patients were included over the study period. The prevalence of CAPA was 5.1% [95% CI 3.4-7.3], and rose to 9.1% among patients who required invasive mechanical ventilation (IMV). Univariable analysis showed that CAPA patients were more frequently immunosuppressed and required more frequently IMV support, vasopressors and renal replacement therapy during ICU stay than non-CAPA patients. SAPS II score at ICU admission, immunosuppression, and a SARS-CoV-2 Delta variant were independently associated with CAPA in multivariable logistic regression analysis. Although CAPA was not significantly associated with day-28 mortality, patients with CAPA experienced a longer duration of mechanical ventilation and ICU stay. CONCLUSION: This study contributes valuable insights into the prevalence, characteristics, and outcomes of CAPA in the era of Delta and Omicron variants. We report a lower prevalence of CAPA (5.1%) among critically-ill COVID-19 patients than previously reported, mainly affecting intubated-patients. Duration of mechanical ventilation and ICU stay were significantly longer in CAPA patients.

Longitudinal analysis of serum neutralization of SARS-CoV-2 Omicron BA.2, BA.4, and BA.5 in patients receiving monoclonal antibodies.

The emergence of Omicron sublineages impacts the therapeutic efficacy of anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) monoclonal antibodies (mAbs). Here, we evaluate neutralization and antibody-dependent cellular cytotoxicity (ADCC) activities of 6 therapeutic mAbs against Delta, BA.2, BA.4, and BA.5. The Omicron subvariants escape most antibodies but remain sensitive to bebtelovimab and cilgavimab. Consistent with their shared spike sequence, BA.4 and BA.5 display identical neutralization profiles. Sotrovimab is the most efficient at eliciting ADCC. We also analyze 121 sera from 40 immunocompromised individuals up to 6 months after infusion of Ronapreve (imdevimab + casirivimab) or Evusheld (cilgavimab + tixagevimab). Sera from Ronapreve-treated individuals do not neutralize Omicron subvariants. Evusheld-treated individuals neutralize BA.2 and BA.5, but titers are reduced. A longitudinal evaluation of sera from Evusheld-treated patients reveals a slow decay of mAb levels and neutralization, which is faster against BA.5. Our data shed light on antiviral activities of therapeutic mAbs and the duration of effectiveness of Evusheld pre-exposure prophylaxis.

Duration of BA.5 neutralization in sera and nasal swabs from SARS-CoV-2 vaccinated individuals, with or without omicron breakthrough infection.

BACKGROUND: Since early 2022, Omicron BA.1 has been eclipsed by BA.2, which was in turn outcompeted by BA.5, which displays enhanced antibody escape properties. METHODS: Here, we evaluated the duration of the neutralizing antibody (Nab) response, up to 18 months after Pfizer BNT162b2 vaccination, in individuals with or without BA.1/BA.2 breakthrough infection. We measured neutralization of the ancestral D614G lineage, Delta, and Omicron BA.1, BA.2, and BA.5 variants in 300 sera and 35 nasal swabs from 27 individuals. FINDINGS: Upon vaccination, serum Nab titers were decreased by 10-, 15-, and 25-fold for BA.1, BA.2, and BA.5, respectively, compared with D614G. We estimated that, after boosting, the duration of neutralization was markedly shortened from 11.5 months with D614G to 5.5 months with BA.5. After breakthrough, we observed a sharp increase of Nabs against Omicron subvariants, followed by a plateau and a slow decline after 5-6 months. In nasal swabs, infection, but not vaccination, triggered a strong immunoglobulin A (IgA) response and a detectable Omicron-neutralizing activity. CONCLUSIONS: BA.5 spread is partly due to abbreviated vaccine efficacy, particularly in individuals who were not infected with previous Omicron variants. FUNDING: Work in O.S.'s laboratory is funded by the Institut Pasteur, Urgence COVID-19 Fundraising Campaign of Institut Pasteur, Fondation pour la Recherche Médicale (FRM), ANRS, the Vaccine Research Institute (ANR-10-LABX-77), Labex IBEID (ANR-10-LABX-62-IBEID), ANR/FRM Flash Covid PROTEO-SARS-CoV-2, ANR Coronamito, and IDISCOVR, Laboratoire d'Excellence 'Integrative Biology of Emerging Infectious Diseases' (grant no. ANR-10-LABX-62-IBEID), HERA european funding and the NIH PICREID (grant no U01AI151758).

Treatment of chronic hepatitis B: virological and pharmacological aspects / Traitement de l'hépatite B chronique par ténofovir : aspects virologiques et pharmacologiques

Tenofovir is a nucleotidic analog inhibitor used in monotherapy as first line treatment of chronic Hepatitis B virus (HBV) infection. This drug requires a two-step phosphorylation by cellular kinases. The active triphosphate form inhibits viral DNA polymerase. Tenofovir has a very low oral bioavailability following oral administration. Hence, its oral administration requires the use of prodrugs: tenofovir disoproxil fumararate (TDF) or tenofovir alafénamide (TAF). TAF demonstrated a lower kidney and bone toxicity than TDF. TAF and TDF treatments allow prevention of chronic hepatitis B complications, which are cirrhosis and hepatocellular carcinoma. Prevention of these complications requires a virological response to the treatment, defined as undetectable HBV DNA. TDF and TAF are associated with virological response rates from 64 to 94 % after one year of treatment. This rate depends on HBV viral load at diagnostic, HBe antigen status, mutations in the HBV polymerase gene (Pol/RT) and patient compliance to the treatment. Tenofovir has a high genetic barrier and resistance mutations to this drug have not yet been described. However, mutations rt181T/V and/or rtN236T have been associated with reduced susceptibility to TDF/TAF in vitro and delayed response in vivo. Recently described mutations CYEI and rtA194T have been associated with reduced susceptibility to TDF/TAF in vitro without any change in viral response in vivo. Patient compliance can be improved using cognitive behavioral therapy, supporter interventions and use of short message service. Finally, some genetic polymorphisms in MRP(multidrug resistance-associated protein)-2 and MRP4 efflux transporters could be associated with TDF toxicity and virological response to TDF or TAF. In the perspective of functional HBV cure, TDF and TAF are likely to be still used, in association with new class of antivirals. For this reason, it is important to further characterize the pharmacological and virological factors associated with partial virological response to tenofovir.

Le ténofovir est un analogue nucléotidique inhibiteur de l'ADN polymérase à activité de transcriptase inverse du virus de l'hépatite B (VHB). Il nécessite une métabolisation intra-cellulaire par des kinases cellulaires pour obtenir la forme triphosphate active. Cette dernière est incorporée à l'ADN viral en cours de synthèse et exerce un effet terminateur de chaîne. Le ténofovir est administré sous forme de promédicaments : le ténofovir disoproxil fumarate (TDF) ou le ténofovir alafénamide (TAF). Le TAF se caractérise par une meilleure tolérance rénale et osseuse que le TDF. Le TDF et le TAF sont indiqués en monothérapie de longue durée pour le traitement de première intention des hépatites B chroniques. Ce traitement permet de prévenir la cirrhose et le carcinome hépatocellulaire qui sont les principales complications de l'hépatite B chronique. Le critère principal de suivi est l'obtention d'une réponse virologique, définie par une charge virale VHB indétectable. Le TDF et le TAF sont associés à des taux de réponse virologique de 64 à 94 % après un an de traitement. Ce taux de réponse dépend de facteurs virologiques, notamment le statut antigène HBe, la charge virale VHB au diagnostic et la présence de certaines mutations dans la polymérase virale (Pol/RT). Aucune résistance au ténofovir n'a été décrite au cours des essais cliniques, ce qui reflète la barrière génétique élevée associée à cette molécule. Néanmoins, les mutations rt181T/V et/ou rtN236T peuvent réduire la sensibilité au ténofovir in vitro et retarder la réponse virologique in vivo. Les mutations CYEI et A194T, récemment décrites, diminuent aussi la sensibilité au ténofovir in vitro mais ne semblent pas avoir d'impact sur la réponse au traitement. La réponse virologique dépend aussi de facteurs de l'hôte, principalement l'observance au traitement, cruciale pour prévenir les complications de l'infection. Cette observance peut être améliorée par des approches comportementales, le soutien d'un tiers ou des rappels par message texte. Enfin, certains polymorphismes génétiques au niveau des transporteurs MRP (multidrug resistance-associated protein)-2 ou MRP4 pourraient moduler la réponse virologique et la tolérance rénale du ténofovir. Dans une perspective de guérison fonctionnelle de l'hépatite chronique B, le ténofovir sera très probablement toujours utilisé, en association avec de nouveaux antiviraux. Pour ces raisons, il semble important de poursuivre l'identification des facteurs pharmacologiques et virologiques associés à la réponse virologique au ténofovir.

Antibody Responses after a Third Dose of COVID-19 Vaccine in Kidney Transplant Recipients and Patients Treated for Chronic Lymphocytic Leukemia.

The impact of a third dose of COVID-19 vaccine on antibody responses is unclear in immunocompromised patients. The objective of this retrospective study was to characterize antibody responses induced by a third dose of mRNA COVID-19 vaccine in 160 kidney transplant recipients and 20 patients treated for chronic lymphocytic leukemia (CLL). Prevalence of anti-spike IgG ≥ 7.1 and ≥ 30 BAU/mL after the third dose were 47% (75/160) and 39% (63/160) in kidney transplant recipients, and 57% (29/51) and 50% (10/20) in patients treated for CLL. Longitudinal follow-up identified a moderate increase in SARS-CoV-2 anti-spike IgG levels after a third dose of vaccine in kidney transplant recipients (0.19 vs. 5.28 BAU/mL, p = 0.03) and in patients treated for CLL (0.63 vs. 10.7 BAU/mL, p = 0.0002). This increase in IgG levels had a limited impact on prevalence of anti-spike IgG ≥ 30 BAU/mL in kidney transplant recipients (17%, 2/12 vs. 33%, 4/12, p = 0.64) and in patients treated for CLL (5%, 1/20 vs. 45%, 9/20, p = 0.008). These results highlight the need for vaccination of the general population and the importance of non-medical preventive measures to protect immunocompromised patients.

Evolution and phenotypic characterization of whole HBV genome in compliant patients experiencing unexplained entecavir treatment failure.

Entecavir treatment failure can be observed in compliant patients despite an absence of detectable resistance mutations by Pol/RT Sanger sequencing. We hypothesized that these unexplained treatment failures could rely on other mechanisms of viral resistance, especially on mutations selected outside of the Pol/RT domain. Partial virological response to entecavir was observed in three patients treated with immunosuppressive drugs, without selection of Pol/RT resistance mutations. Mutations selected in the whole HBV genome during entecavir treatment and potentially associated with resistance were searched for using deep sequencing and characterized using a phenotypic resistance assay. Mutations Q206K (pre-core/core), Q120K (pre-S1/pre-S2, T-cell epitope) and A300E (spacer domain) were selected during entecavir treatment in patient #1 but were not associated with an increased level of resistance to entecavir or an increase in HBV replication capacity. Core promoter mutations T1753G, A1762T and G1764A were present as major mutations before and after treatment in patient #1. HBs Ag immune escape mutations were present as major mutations before and after treatment in patients #2 (sK122R, sT126I, sP127S and sG145R) and #3 (sM133I). We demonstrated that PVR to entecavir does not require selection of any resistance mutation in the whole HBV genome. Our results demonstrate that major mutations can be selected outside of the Pol/RT domain before or during entecavir treatment. These mutations could contribute to entecavir treatment failure by other mechanisms than an increased level of resistance.

[Viruses : vesicle tamers]. / Les virus : des dompteurs de vésicules.

There is a close relationship between viruses and lipid vesicles. The most frequently described concerns enveloped viruses, which acquire their envelope through mechanisms involved in extracellular vesicles (EVs) biogenesis. However, EVs' hijacking is not unique to enveloped viruses. In 2013, a new category of viruses emerged : the quasi-enveloped viruses. These are naked viruses found in vesicles at certain steps of their viral cycle. Actually, several naked viruses, from different families, hijack the production routes of EVs : poliovirus, polyomaviruses, rotavirus, etc. This diversion of EVs confers many advantages : diversification of entry and exit pathways, infectivity improvement and immune evasion. This review will take the reader around this subject.

Intercellular Transmission of Naked Viruses through Extracellular Vesicles: Focus on Polyomaviruses.

Extracellular vesicles have recently emerged as a novel mode of viral transmission exploited by naked viruses to exit host cells through a nonlytic pathway. Extracellular vesicles can allow multiple viral particles to collectively traffic in and out of cells, thus enhancing the viral fitness and diversifying the transmission routes while evading the immune system. This has been shown for several RNA viruses that belong to the Picornaviridae, Hepeviridae, Reoviridae, and Caliciviridae families; however, recent studies also demonstrated that the BK and JC viruses, two DNA viruses that belong to the Polyomaviridae family, use a similar strategy. In this review, we provide an update on recent advances in understanding the mechanisms used by naked viruses to hijack extracellular vesicles, and we discuss the implications for the biology of polyomaviruses.

Indoleamine 2,3-Dioxygenase Is Involved in Interferon Gamma's Anti-BKPyV Activity in Renal Cells.

Reactivation of BK polyomavirus (BKPyV) infection is frequently increasing in transplant recipients treated with potent immunosuppressants and highlights the importance of immune system components in controlling viral reactivation. However, the immune response to BKPyV in general and the role of antiviral cytokines in infection control in particular are poorly understood. Here, we investigated the efficacy of interferons (IFN) alpha, lambda and gamma with regard to the BKPyV multiplication in Vero cells. Treatment with IFN-gamma inhibited the expression of the viral protein VP1 in a dose-dependent manner and decreased the expression of early and late viral transcripts. Viral inhibition by IFN-gamma was confirmed in human cells (Caki-1 cells and renal proximal tubular epithelial cells). One of the IFN-stimulated genes most strongly induced by IFN-gamma was the coding for the enzyme indoleamine 2,3 dioxygenase (IDO), which is known to limit viral replication and regulates the host immune system. The antiviral activity induced by IFN-gamma could be reversed by the addition of an IDO inhibitor, indicating that IDO has a specific role in anti-BKPyV activity. A better understanding of the action mechanism of these IFN-gamma-induced antiviral proteins might facilitate the development of novel therapeutic strategies.

Comparison of different serological assays for SARS-CoV-2 in real life.

BACKGROUND: The emergence of the global SARS-CoV-2 pandemic required the rapid and large-scale deployment of PCR and serological tests in different formats. OBJECTIVES: Real-life evaluation of these tests is needed. Using 168 samples from patients hospitalized for COVID-19, non-hospitalized patients but infected with SARS-CoV-2, patients participating in screening campaigns, and samples from patients with a history of other seasonal coronavirus infections, we evaluated the clinical performance of 5 serological assays widely used worldwide (WANTAI®, BIORAD®, EUROIMMUN®, ABBOTT® and LIAISON®). RESULTS: For hospitalized patients, all these assays showed a sensitivity of 100 % from day 9 after the symptoms onset. On the other hand, sensitivity was much lower for patients who did not require hospitalization for COVID-19 confirmed by PCR (from 91.6 % for WANTAI® to 69 % for LIAISON®). These differences do not seem to be due to the antigens chosen by the manufacturers but more to the test formats (IgG detection versus total antibodies). In addition, more than 50 days after a positive PCR for CoV-2-SARS the proportion of positive patients seem to decrease. We did not observe any significant cross-reactions for these techniques with the four other seasonal coronaviruses. CONCLUSION: In conclusion, the evaluation and knowledge of the serological tests used is important and should require an optimized strategy adaptation of the analysis laboratories to best meet patient's expectations in the face of this health crisis.

BK Polyomavirus Hijacks Extracellular Vesicles for En Bloc Transmission.

Most people are asymptomatic carriers of the BK polyomavirus (BKPyV), but the mechanisms of persistence and immune evasion remain poorly understood. Furthermore, BKPyV is responsible for nephropathies in kidney transplant recipients. Unfortunately, the sole therapeutic option is to modulate immunosuppression, which increases the risk of transplant rejection. Using iodixanol density gradients, we observed that Vero and renal proximal tubular epithelial infected cells release two populations of infectious particles, one of which cosediments with extracellular vesicles (EVs). Electron microscopy confirmed that a single vesicle could traffic tens of viral particles. In contrast to naked virions, the EV-associated particles (eBKPyVs) were not able to agglutinate red blood cells and did not use cell surface sialylated glycans as an attachment factor, demonstrating that different entry pathways were involved for each type of infectious particle. However, we also observed that naked BKPyV and eBKPyV were equally sensitive to neutralization by the serum of a seropositive patient or commercially available polyvalent immunoglobulin preparations, which occurred at a postattachment step, after endocytosis. In conclusion, our work shows a new mechanism that likely plays a critical role during the primary infection and in the persistence, but also the reactivation, of BKPyV.IMPORTANCE Reactivation of BKPyV is responsible for nephropathies in kidney transplant recipients, which frequently lead to graft loss. The mechanisms of persistence and immune evasion used by this virus remain poorly understood, and a therapeutic option for transplant patients is still lacking. Here, we show that BKPyV can be released into EVs, enabling viral particles to infect cells using an alternative entry pathway. This provides a new view of BKPyV pathogenesis. Even though we did not find any decreased sensitivity to neutralizing antibodies when comparing EV-associated particles and naked virions, our study also raises important questions about developing prevention strategies based on the induction or administration of neutralizing antibodies. Deciphering this new release pathway could enable the identification of therapeutic targets to prevent BKPyV nephropathies. It could also lead to a better understanding of the pathophysiology of other polyomaviruses that are associated with human diseases.

No correlation between Torque Teno virus viral load and BK virus replication after kidney transplantation.

BACKGROUND: Assessment of the intensity of immunosuppression in transplant recipients to estimate the risk of rejection and infection is not entirely satisfactory at the present time. Determination of Torque teno virus (TTV) viral load appears to be a promising tool in this setting. OBJECTIVES: We evaluated the level of replication and kinetics of TTV during the first three months after kidney transplantation compared to BK virus replication. RESULTS: In a retrospective cohort of 116 renal transplant recipients, TTV viral load gradually increased during the first three months post-transplantation with no significant difference or discriminatory threshold between patients with and without BK virus replication. However, the level of TTV replication appeared to be indirectly related to the risk of BK virus replication, particularly according to the induction treatment used (antithymocyte globulin: ATG or basiliximab). Among patients receiving ATG, those receiving cyclosporine had significantly lower TTV viral loads (p < 0.01) with threefold lower reactivation of BKPyV (13 vs 37%) 3 months post-transplantation. Similarly, among the women in our cohort, TTV viral load was significantly higher in women receiving ATG (6.58 ± 1.57 versus 4.62 ± 2.0 log10 copies/mL for basiliximab: p < 0.01), also with threefold higher BKPyV reactivation frequencies (40 vs 13,3%). CONCLUSION: The multiparametric variation of TTV viral load does not appear to be individually appropriate for the early detection or monitoring of possible post-transplant BKPyV virus reactivation in renal transplant recipients.

QuantIF: An ImageJ Macro to Automatically Determine the Percentage of Infected Cells after Immunofluorescence.

Counting labeled cells, after immunofluorescence or expression of a genetically fluorescent reporter protein, is frequently used to quantify viral infection. However, this can be very tedious without a high content screening apparatus. For this reason, we have developed QuantIF, an ImageJ macro that automatically determines the total number of cells and the number of labeled cells from two images of the same field, using DAPI- and specific-stainings, respectively. QuantIF can automatically analyze hundreds of images, taking approximately one second for each field. It is freely available as supplementary data online at MDPI.com and has been developed using ImageJ, a free image processing program that can run on any computer with a Java virtual machine, which is distributed for Windows, Mac, and Linux. It is routinely used in our labs to quantify viral infections in vitro, but can easily be used for other applications that require quantification of labeled cells.

Biology of the BKPyV: An Update.

The BK virus (BKPyV) is a member of the Polyomaviridae family first isolated in 1971. BKPyV causes frequent infections during childhood and establishes persistent infections with minimal clinical implications within renal tubular cells and the urothelium. However, reactivation of BKPyV in immunocompromised individuals may cause serious complications. In particular, with the implementation of more potent immunosuppressive drugs in the last decade, BKPyV has become an emerging pathogen in kidney and bone marrow transplant recipients where it often causes associated nephropathy and haemorrhagic cystitis, respectively. Unfortunately, no specific antiviral against BKPyV has been approved yet and the only therapeutic option is a modulation of the immunosuppressive drug regimen to improve immune control though it may increase the risk of rejection. A better understanding of the BKPyV life cycle is thus needed to develop efficient treatment against this virus. In this review, we provide an update on recent advances in understanding the biology of BKPyV.

DHEA prevents ribavirin-induced anemia via inhibition of glucose-6-phosphate dehydrogenase.

Ribavirin has been widely used for antiviral therapy. Unfortunately, ribavirin-induced anemia is often a cause of limiting or interrupting treatment. Our team has observed that dehydroepiandrosterone (DHEA) has a protective effect against in vitro and in vivo ribavirin-induced hemolysis. The aim of this study was to better understand this effect as well as the underlying mechanism(s). DHEA was able to reduce in vitro intraerythrocytic ATP depletion induced by ribavirin. Only 1% of ATP remained after incubation with ribavirin (2 mM) at 37 °C for 24 h vs. 37% if DHEA (200 µM) was added (p < 0.01). DHEA also helped erythrocytes conserve their size, with a shrinkage of only 10% vs 40% at 24 h with ribavirin alone (p < 0.01), and reduced phosphatidylserine exposure at the outer membrane, i.e. 27% vs 40% at 48 h, (p < 0.05). DHEA also inhibits ribavirin-induced hemolysis, i.e. 34% vs 46.5% at 72 h (p < 0.01). DHEA is an inhibitor of glucose-6-phosphate dehydrogenase (G6PD), a key enzyme in the hexose monophosphate shunt connected to the glycolytic pathway which is the only energy supplier of the red blood cell in the form of ATP. We have confirmed this inhibitory effect in the presence of ribavirin. All these observations suggest that ribavirin-induced hemolysis was initiated by ATP depletion, and that the inhibitory effect of DHEA on G6PD was able to rescue enough ATP to limit this hemolysis. This mechanism could be important for improving the therapeutic management of patients treated with ribavirin.