Validation of a SARS-CoV-2 Surrogate Virus Neutralization Test in Recovered and Vaccinated Healthcare Workers.

Vaccination against COVID-19 is the main public health approach to fight against the pandemic. The Spike (S) glycoprotein of SARS-CoV-2 is the principal target of the neutralizing humoral response. We evaluated the analytical and clinical performances of a surrogate virus neutralization test (sVNT) compared to conventional neutralization tests (cVNTs) and anti-S eCLIA assays in recovered and/or vaccinated healthcare workers. Our results indicate that sVNTs displayed high specificity and no cross-reactivity. Both eCLIA and sVNT immunoassays were good at identifying cVNT serum dilutions ≥1:16. The optimal thresholds when identifying cVNT titers ≥1:16, were 74.5 U/mL and 49.4 IU/mL for anti-S eCLIA and sVNT, respectively. Our data show that neutralizing antibody titers (Nab) differ from one individual to another and may diminish over time. Specific assays such as sVNTs could offer a reliable complementary tool to routine anti-S serological assays.

Persistence of Neutralizing Antibodies and Clinical Protection up to 12†Months After Severe Acute Respiratory Syndrome Coronavirus 2 Infection in the Elderly.

Background: Coronavirus disease 2019 (COVID-19) has severely affected the elderly, who are expected to display decreased immune responses due to immunosenescence. Methods: This study retrospectively assesses neutralizing antibody (NAb) production up to 12 months after infection in long-term care patients. We used Roche Diagnostics immunoassay to quantify anti-spike (S) antibodies and a competitive immunoassay from YHLO as a surrogate test for NAb. Results: We included 91 patients (mean age, 86 years). There was no significant variation in anti-S titers over time. There was a significant decrease of NAb titers between month 3 and month 6 but no further significant change up to month 12. Overall, 75 of 91 (82%) and 52 of 91 (57%) patients had, at least once, anti-S titers >75 U/mL and NAb titers >50 AU/mL, respectively, corresponding to a significant neutralizing activity in vitro. All 68 patients studied at M12 had detectable anti-S antibodies and 60 (88%) had detectable NAb; 60 of 68 (88%) and 29 of 68 (42.6%) still had anti-S titers >75 U/mL and NAb titers >50 AU/mL. Higher NAb titers were correlated with severe infection, higher levels of C-reactive protein, and lower lymphocyte counts. No patient developed reinfection. Conclusions: Elderly people can display robust and persistent humoral response after severe acute respiratory syndrome coronavirus 2 infection, with NAb lasting up to 12 months.

Contribution of Serum Cytomegalovirus PCR to Diagnosis of Early CMV Primary Infection in Pregnant Women.

(1) Background: What is the role of serum CMV PCR in the diagnosis of recent primary infection (PI) in pregnant women when IgG avidity is uninformative? (2) Methods: Retrospective cohort study to compare serum versus whole blood CMV PCR. (a) Qualitative assessment: CMV PCR was performed on 123 serum samples and 74 whole blood samples collected from 132 pregnant women with recent CMV PI. PCR positivity rate was used to calculate sensitivity in serum and whole blood. (b) Quantitative assessment: CMV PCR was performed on 72 paired samples of serum and whole blood collected on the same day from 57 patients. (3) Results: In pregnant women, PCR positivity rate was 89% for serum samples versus 100% in whole blood in the case of very recent PI (<15 days), but only 27% in serum versus 68% in whole blood for PI occurring from 6 weeks to 3 months before. Comparing CMV viral loads between serum and whole blood, we determined the limit of CMV DNA detection in serum as 3 log copies/mL (whole blood equivalent). (4) Conclusions: Serum CMV PCR is reliable in confirming PI in cases when only IgM is detected. It is therefore a valuable tool in introducing valaciclovir treatment as early as possible to prevent mother-to-child CMV transmission.

Transfusion-Transmitted Hepatitis A Virus, France, 2018.

We report a transfusion-transmitted hepatitis A virus infection in an immunocompromised patient in France, detected shortly after a transfusion of pathogen-reduced pooled platelets. This case raises questions about the efficacy of donor screening methods. Additional safety measures, such as routine donation screening, should be considered.

Rituximab Impairs B Cell Response But Not T Cell Response to COVID-19 Vaccine in Autoimmune Diseases.

OBJECTIVE: Antibody response to the messenger RNA (mRNA) COVID-19 vaccine has been shown to be diminished in rituximab (RTX)-treated patients. We undertook this study to compare humoral and T cell responses between healthy controls, patients with autoimmune diseases treated with RTX, and those treated with other immunosuppressants, all of whom had been vaccinated with 2 doses of the mRNA COVID-19 vaccine. METHODS: We performed anti-spike IgG and neutralization assays just before and 28 days after the second BNT162b2 (Pfizer-BioNTech) vaccine dose. The specific T cell response was assessed in activated CD4 and CD8 T cells using intracellular flow cytometry staining of cytokines (interferon-γ, tumor necrosis factor, and interleukin-2) after stimulation with SARS-CoV-2 spike peptide pools. RESULTS: A lower proportion of responders with neutralizing antibodies to the vaccine was observed in the RTX group (29%; n = 24) compared to the other immunosuppressants group (80%; n = 35) (P = 0.0001) and the healthy control group (92%; n = 26) (P < 0.0001). No patients treated with RTX in the last 6 months showed a response. Time since last infusion was the main factor influencing humoral response in RTX-treated patients. The functional CD4 and CD8 cellular responses to SARS-CoV-2 peptides for each single cytokine or polyfunctionality were not different in the RTX group compared to the other immunosuppressants group or the control group. In RTX-treated patients, the T cell response was not different between patients with and those without a humoral response. CONCLUSION: RTX induced a diminished antibody response to the mRNA COVID-19 vaccine, but the functional T cell response was not altered compared to healthy controls and autoimmune disease patients treated with other immunosuppressants. Further work is needed to assess the clinical protection granted by a functionally active T cell response in the absence of an anti-spike antibody response.

Accuracy assessment of total or IgG Immunoglobulin to hepatitis A virus tests around immunity threshold.

BACKGROUND: Anti-hepatitis A virus (HAV) antibody titers at 20 IU/L are assumed to correlate with protection against HAV challenge. METHODS: We examined the accuracy and precision of currently in use immunoassays for total or anti-HAV IgG determination, by repeated testing of dilutions of the international anti-HAV standard, within a 10-50 IU/mL concentration range. RESULTS AND CONCLUSION: Eight immunoassays were evaluated. All could confidently identify people who need to be vaccinated, or who might benefit from a booster vaccine: no positive interpretation for the 10 and 15 IU/mL concentrations. However, qualitative interpretation may differ from test to test in the 15-30 IU/mL range. This variation has to be taken into account when comparing seroprevalence data.

Emergence of SARS-CoV-2 resistance mutations in a patient who received anti-SARS-COV2 spike protein monoclonal antibodies: a case report.

BACKGROUND: To manage severe or potentially severe cases of CoronaVirus Disease 2019 (COVID-19), therapeutic monoclonal antibodies targeting Spike protein of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) have been designed. It has been noted in vitro that upon exposure to these treatments, mutations could be selected. CASE PRESENTATION: We here report the case of an immunosuppressed patient infected with a B.1.1.7 variant, who received a combination of monoclonal antibodies, and subsequently selected mutations K417N, E484K and Q493R on Spike protein of SARS-CoV-2. CONCLUSIONS: Our case raises the importance of monitoring SARS-CoV-2 mutations in patients receiving monoclonal antibodies and having persistent excretion of the virus, in order to offer optimal management of their infection, and strengthen prevention measures to avoid subsequent transmission of these selected variants.

Response to COVID-19 mRNA vaccination in multiple myeloma is conserved but impaired compared to controls.

Patients with multiple myeloma are at high risk of severe forms of COVID-19. Despite data showing diminished response to vaccine, the era of highly efficient mRNA vaccine might be a gamechanger. We sought to examine response to mRNA vaccine between healthy controls (n = 28) and multiple myeloma (MM) patients (n = 27). Response was analyzed 1 month after the second dose of anti-SARS-CoV-2 BNT162b2 vaccine. Multiple myeloma patients showed diminished levels of Anti-Spike IgG levels compared to controls, but with a high proportion of patients achieving a humoral response (89% vs. 97% in controls). Neutralizing antibodies were present in 74% of patients versus 96% of controls. Patients under current daratumumab treatment had neutralizing activity of anti-SARS-CoV-2 antibodies. Multiple myeloma patients show diminished response to SARS-COV-2 vaccine but with still high response rate. The main potential risk factor of non-response to COVID-19 vaccine was uncontrolled disease under treatment.

BHRF1, a BCL2 viral homolog, disturbs mitochondrial dynamics and stimulates mitophagy to dampen type I IFN induction.

Mitochondria respond to many cellular functions and act as central hubs in innate immunity against viruses. This response is notably due to their role in the activation of interferon (IFN) signaling pathways through the activity of MAVS (mitochondrial antiviral signaling protein) present at the mitochondrial surface. Here, we report that the BHRF1 protein, a BCL2 homolog encoded by Epstein-Barr virus (EBV), inhibits IFNB/IFN-ß induction by targeting the mitochondria. Indeed, we have demonstrated that BHRF1 expression modifies mitochondrial dynamics and stimulates DNM1L/Drp1-mediated mitochondrial fission. Concomitantly, we have shown that BHRF1 is pro-autophagic because it stimulates the autophagic flux by interacting with BECN1/Beclin 1. In response to the BHRF1-induced mitochondrial fission and macroautophagy/autophagy stimulation, BHRF1 drives mitochondrial network reorganization to form juxtanuclear mitochondrial aggregates known as mito-aggresomes. Mitophagy is a cellular process, which can specifically sequester and degrade mitochondria. Our confocal studies uncovered that numerous mitochondria are present in autophagosomes and acidic compartments using BHRF1-expressing cells. Moreover, mito-aggresome formation allows the induction of mitophagy and the accumulation of PINK1 at the mitochondria. As BHRF1 modulates the mitochondrial fate, we explored the effect of BHRF1 on innate immunity and showed that BHRF1 expression could prevent IFNB induction. Indeed, BHRF1 inhibits the IFNB promoter activation and blocks the nuclear translocation of IRF3 (interferon regulatory factor 3). Thus, we concluded that BHRF1 can counteract innate immunity activation by inducing fission of the mitochondria to facilitate their sequestration in mitophagosomes for degradation.Abbreviations: 3-MA: 3-methyladenine; ACTB: actin beta; BCL2: BCL2 apoptosis regulator; CARD: caspase recruitment domain; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; CI: compaction index; CQ: chloroquine; DAPI: 4',6-diamidino-2-phenylindole, dihydrochloride; DDX58/RIG-I: DExD/H-box helicase 58; DNM1L/Drp1: dynamin 1 like; EBSS: Earle's balanced salt solution; EBV: Epstein-Barr virus; ER: endoplasmic reticulum; EV: empty vector; GFP: green fluorescent protein; HEK: human embryonic kidney; IFN: interferon; IgG: immunoglobulin G; IRF3: interferon regulatory factor 3; LDHA: lactate dehydrogenase A; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAVS: mitochondrial antiviral signaling protein; MMP: mitochondrial membrane potential; MOM: mitochondrial outer membrane; PINK1: PTEN induced kinase 1; RFP: red fluorescent protein; ROS: reactive oxygen species; SQSTM1/p62: sequestosome 1; STING1: stimulator of interferon response cGAMP interactor 1; TOMM20: translocase of outer mitochondrial membrane 20; VDAC: voltage dependent anion channel.

Comparison of the Abbott Alinity m and m2000 assays for the quantification of HIV-1, HCV and HBV in clinical samples.

BACKGROUND: Viral load (VL) determination is an essential parameter of the management of patients infected with HIV, HBV or HCV. Many available molecular systems run on a "batch" mode while "random access" systems provide more flexibility. OBJECTIVES: We compared the performance of HIV-1, HCV and HBV quantification assays on the recently developed Abbott Alinity m system to the m2000 RealTime assays. STUDY DESIGN: Plasma specimens sent for viral load determination were prospectively tested on m2000 and Alinity m systems, according to manufacturers' instructions. Additional low and high tittered samples were used to assess reproducibility. RESULTS: Assays concordance was evaluated from 180 samples for HIV-1, 122 for HBV, and 92 for HCV. A good correlation and a linear relation over the quantification range was observed for the three markers (r > 0.974). The Alinity m assays yielded higher results with a mean quantification bias of 0.22 log cp/ml for 75 HIV-1, 0.3 log IU/ml for 79 HBV, and 0.2 log for 35 HCV samples, though results were equivalent within an allowable difference of 0.3-0.4 log. Qualitative discordance was observed for 43/180 HIV results, 10/122 HBV and 7/92 HCV and involved undetectable or low-level VL. CONCLUSION: The Alinity m assays have performance equivalent to m2000. Upon implementation, physicians should be aware of the relative overquantification compared to previous Abbott assays, particularly around clinical decision thresholds. With reduced turnarounds and hands-on times compared to the m2000 system, the Alinity m platform may improve significantly the laboratory workflow efficiency for the benefit of physicians and patients.

Invasive meningoencephalitis as the first manifestation of hepatitis A.

Since mid-2016, Europe has been facing a major hepatitis A epidemic, with more than 25 000 cases reported to the European CDC, in 2018. We describe herein a rare case of invasive HAV meningoencephalitis as a prodrome of the classic hepatitis, which largely explains a delay in diagnosis.

Transfusion-Transmitted Hepatitis E Virus Infection in France.

There is growing concern regarding the risk of transfusion- transmitted (TT) hepatitis E. Since the first described case in 2006, several TT hepatitis E have been reported to the French hemovigilance network. We performed a retrospective analysis of all cases of TT hepatitis E reported between 2006 and 2016. Transfusion-transmitted hepatitis E with high imputability according to phylogenetic analysis occurred in 23 patients aged 8 to 88 years and involved mostly solid organ recipients (n = 9) or patients with malignant hematological diseases (n = 9, including 4 hematopoietic allograft recipients). Involved blood products were plasma (n = 7), among which 6 had undergone pathogen reduction with solvent/detergent (n = 4) or amotosalen + ultra-violet A (UVA) (n = 2 from 1 donation) treatments, red blood concentrates (n = 7), apheresis platelets concentrates (n = 3) and whole blood pooled platelets concentrates (n = 6), among which one had underwent amotosalen + UVA treatment. Median hepatitis E virus (HEV) RNA dose infused was 5.79 [4.36-10.10] log IU. HEV infection progressed to chronic hepatitis E in 14 (61%) immunocompromised patients, 2 of whom had advanced liver fibrosis at diagnosis. Chronic hepatitis E patients cleared HEV with ribavirin treatment (n = 10), after immunosuppressive drug reduction (n = 3), or spontaneously (n = 1). One additional organ transplant recipient with associated co-morbidities died with ongoing HEV infection and multiple organ failure. The other 8 (34.8%) patients with TT hepatitis E cleared HEV within 6 months with ribavirin treatment (n = 3), reduced immunosuppression (n = 1) or spontaneously (n = 4). Red cells, platelets, and plasma transfusions may be associated with TT hepatitis E that can evolve to chronic hepatitis E in immunocompromised patients. Hepatitis E virus has emerged in France as a clinically significant TT infection risk.

Human cytomegalovirus hijacks the autophagic machinery and LC3 homologs in order to optimize cytoplasmic envelopment of mature infectious particles.

During its life cycle, Human cytomegalovirus (HCMV) tightly modulates autophagy, a vesicular pathway allowing degradation and recycling of cellular components. To study the interplay between autophagy and the viral life cycle, we established various autophagy-deficient human fibroblastic cell lines. By knocking down the expression or activity of five autophagy-related proteins, we confirmed the proviral function that the autophagic machinery exerts on HCMV production. Using 3D reconstruction from confocal microscopy and electron microscopy, we demonstrated that lipidated LC3-positive vesicles accumulated at the viral assembly compartment (vAC). The vAC is a juxtanuclear ring-shaped structure containing several organelles and membranes, where assembly and final envelopment of HCMV particles occur. Two LC3 homologs, GABARAPL1 and GATE16, also accumulated during HCMV infection and were associated with the vAC, in proximity with fragmented Golgi stacks. Additionally, we observed the formation of a pre-assembly compartment (PrAC) in infected cells, which consists of a juxtanuclear structure containing both fragmented Golgi and LC3-positive vesicles. Finally, we showed that highly purified extracellular viral particles were associated with various autophagy proteins. Our results thus suggest that autophagy machinery participates to the final cytoplasmic envelopment of HCMV viral particles into the vAC and that autophagy-related proteins can be spotted in the virions.

Two concurrent outbreaks of hepatitis A highlight the risk of infection for non-immune travellers to Morocco, January to June 2018.

From January to June 2018, two ongoing hepatitis A outbreaks affected travellers returning from Morocco and cases in Europe without travel history, resulting in 163 patients in eight European countries. Most interviewed travel-related cases were unaware of the hepatitis A risk in Morocco. Molecular analysis revealed two distinct hepatitis A virus (HAV) strains (subgenotype IA DK2018_231; subgenotype IB V18-16428). Vaccination recommendations should be emphasised to increase awareness among non-immune travellers to Morocco and HAV-endemic countries.

Hepatitis A: an epidemiological survey in blood donors, France 2015 to 2017.

Since mid-2016, hepatitis A virus (HAV) outbreaks, involving predominantly men who have sex with men (MSM), have affected countries in Europe and overseas. In France, HAV screening of blood donations in 2017 revealed a HAV-RNA prevalence ca fivefold higher than during 2015-16 (4.42/106 vs 0.86/106; p = 0.0005). In 2017, despite a higher male-to-female ratio (5.5 vs 0.7) and the identification of MSM-associated outbreak strains, only one of 11 infected male donors self-reported being a MSM.

Analysis of the role of autophagy inhibition by two complementary human cytomegalovirus BECN1/Beclin 1-binding proteins.

Autophagy is activated early after human cytomegalovirus (HCMV) infection but, later on, the virus blocks autophagy. Here we characterized 2 HCMV proteins, TRS1 and IRS1, which inhibit autophagy during infection. Expression of either TRS1 or IRS1 was able to block autophagy in different cell lines, independently of the EIF2S1 kinase, EIF2AK2/PKR. Instead, TRS1 and IRS1 interacted with the autophagy protein BECN1/Beclin 1. We mapped the BECN1-binding domain (BBD) of IRS1 and TRS1 and found it to be essential for autophagy inhibition. Mutant viruses that express only IRS1 or TRS1 partially controlled autophagy, whereas a double mutant virus expressing neither protein stimulated autophagy. A mutant virus that did not express IRS1 and expressed a truncated form of TRS1 in which the BBD was deleted, failed to control autophagy. However, this mutant virus had similar replication kinetics as wild-type virus, suggesting that autophagy inhibition is not critical for viral replication. In fact, using pharmacological modulators of autophagy and inhibition of autophagy by shRNA knockdown, we discovered that stimulating autophagy enhanced viral replication. Conversely, inhibiting autophagy decreased HCMV infection. Thus, our results demonstrate a new proviral role of autophagy for a DNA virus.