Julien Marlet
email: julien.marlet@univ-tours.fr

Emmanuelle Roch

Clément Lier

Alain Moreau

Benjamin Combe

Lynda Handala

Sandrine Lefeuvre

Morgan Maugey

Laure Elkrief

Louis D'alteroche

Louis D'alteroche

Pascal Potier

Denys Brand

Catherine Gaudy-Graffin

Evolution and phenotypic characterization of HBV quasispecies in compliant patients experiencing unexplained entecavir treatment failure Running title: Entecavir, viral resistance & whole genome

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Chronic hepatitis B virus infection affects 292 million people worldwide and is associated with an increase in the risk of cirrhosis and hepatocellular carcinoma (HCC) (Razavi-Shearer et al., 2018). Nucleoside or nucleotide analogs are used as a first-line treatment to prevent these complications [START_REF] Li | Tenofovir versus entecavir in lowering the risk of hepatocellular carcinoma development in patients with chronic hepatitis B: a critical systematic review and meta-analysis[END_REF][START_REF] Papatheodoridis | Hepatocellular carcinoma prediction beyond year 5 of oral therapy in a large cohort of Caucasian patients with chronic hepatitis B[END_REF][START_REF] Xu | Long-term antiviral efficacy of entecavir and liver histology improvement in Chinese patients with hepatitis B virus-related cirrhosis[END_REF]. Two of these molecules, entecavir and tenofovir (disoproxil fumarate or alafenamide) have been associated with higher rates of viral response and a higher drug genetic barrier than previous antiviral drugs (lamivudine, adefovir). Entecavir resistance mutations in the HBV Pol/RT domain are rarely selected (< 1.2%, after five years of treatment) [START_REF] Tenney | Long-term monitoring shows hepatitis B virus resistance to entecavir in nucleoside-naïve patients is rare through 5 years of therapy[END_REF] except in patients with a history of lamivudine treatment [START_REF] Hayashi | Characterization of novel entecavir resistance mutations[END_REF]L. Liu et al., 2019;Y. Liu et al., 2019;[START_REF] Rose | Discovery of the Novel Entecavir-Resistant Hepatitis B Virus Reverse Transcriptase A181C Substitution From an Integrated Genotypic Analysis[END_REF]. These mutations have been associated with primary non-response and viral breakthrough on entecavir treatment, but not with a partial virological response (PVR) to entecavir [START_REF] Lampertico | EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection[END_REF]. This last form of treatment failure is defined as a decrease in HBV DNA levels of more than 1 log10 IU/mL, with HBV DNA detectable after at least 12 months of treatment in compliant patients [START_REF] Lampertico | EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection[END_REF]. It occurs in 10-33% of patients treated with entecavir (Chang et al., 2006;Lai et al., 2006) and has been associated with HBe Ag positivity, and with low levels of ALAT and a high HBV DNA viral load at baseline [START_REF] Chen | Antiviral effect of entecavir in nucleos(t)ide analogue-naïve and nucleos(t)ide analogue-experienced chronic hepatitis B patients without virological response at week 24 or 48 of therapy[END_REF][START_REF] Lee | Prolonged Entecavir Therapy Is Not Effective for HBeAg Seroconversion in Treatment-Naive Chronic Hepatitis B Patients with a Partial Virological Response[END_REF][START_REF] Li | Partial virological response to entecavir treatment in nucleos(t)ide-naïve patients with HBeAg-positive chronic hepatitis B is not caused by reduced sensitivity[END_REF][START_REF] Zoutendijk | Entecavir treatment for chronic hepatitis B: adaptation is not needed for the majority of naïve patients with a partial virological response[END_REF].

The management of patients experiencing entecavir treatment failure is based on the identification of known resistance mutations by Pol/RT Sanger sequencing [START_REF] Lampertico | EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection[END_REF][START_REF] Lok | Antiviral drug-resistant HBV: Standardization of nomenclature and assays and recommendations for management[END_REF] and, if relevant, the characterization of new resistance mutations in phenotypic assays [START_REF] Zoulim | Optimal management of chronic hepatitis B patients with treatment failure and antiviral drug resistance[END_REF]. However, this conventional strategy has several limitations. First, it does not take into account polymorphisms selected outside of the Pol/RT domain and associated with different clinical phenotypes of chronic hepatitis B [START_REF] Huang | Impact of Genetic Heterogeneity in Polymerase of Hepatitis B Virus on Dynamics of Viral Load and Hepatitis B Progression[END_REF][START_REF] Peng | The nucleotide changes within HBV core promoter/precore during the first 12weeks of nucleos(t)ide treatment might be associated with a better virological response[END_REF][START_REF] Podlaha | Large-scale viral genome analysis identifies novel clinical associations between hepatitis B virus and chronically infected patients[END_REF]. Second, unlike deep sequencing, it is unsuitable for the sensitive detection of minor variants before [START_REF] Chevaliez | Primary resistance of hepatitis B virus to nucleoside and nucleotide analogues[END_REF] or after treatment failure [START_REF] Lee | Pyrosequencing method for sensitive detection of HBV drug resistance mutations[END_REF][START_REF] Lowe | Implementation of Next-Generation Sequencing for Hepatitis B Virus Resistance Testing and Genotyping in a Clinical Microbiology Laboratory[END_REF]. This conventional approach fails to detect any Pol/RT resistance mutations in some patients experiencing treatment failure on entecavir. We hypothesized that a more thorough exploration, based on deep sequencing of the whole HBV genome before and after treatment failure, would provide a more comprehensive overview of the viral factors associated with treatment failure.

We applied this strategy to compliant patients experiencing entecavir treatment failure without the selection of mutations of the HBV Pol/RT domain. Relevant mutations selected during entecavir treatment were characterized in a phenotypic resistance assay, based on whole HBV genome sequences from clinical isolates.

MATERIALS AND METHODS

Patients and samples

Plasma samples were collected between 2011 and 2018, at Tours University Hospital, for the diagnosis or follow-up of HBV infection [START_REF] Marlet | Revisiting HBV resistance to entecavir with a phenotypic approach[END_REF]. We included patients experiencing entecavir treatment failure with no detectable Pol/RT resistance mutations [START_REF] Lampertico | EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection[END_REF]. HBV infection and liver disease were followed, and therapeutic drug monitoring was performed, as previously described [START_REF] Marlet | Revisiting HBV resistance to entecavir with a phenotypic approach[END_REF]. This study was approved by the Tours University ethics committee (no. 2019 055).

Genotypic drug resistance testing

Genotypic drug resistance testing was performed by amplifying the Pol/RT domain (aa rt1-rt344) and subjecting it to Sanger sequencing , as previously described [START_REF] Villeneuve | Selection of a hepatitis B virus strain resistant to adefovir in a liver transplantation patient[END_REF].

We searched for resistance mutations as previously described [START_REF] Marlet | Revisiting HBV resistance to entecavir with a phenotypic approach[END_REF] and interpreted them in accordance with the latest EASL guidelines [START_REF] Lampertico | EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection[END_REF].

Deep sequencing of the whole HBV genome

Two overlapping regions of the HBV genome were amplified by PCR with primer P1 (CCGGAAAGCTTGAGCTCTTCTTTTTCACCTCTGCCTAATCA) plus primer 786R (GGACTCAAGATGTTGTACAGACTTGGCC), and with primer P2

(CCGGAAAGCTTGAGCTCTTCAAAAAGTTGCATGGTGCTGG) plus primer BB (CTGGATGTGTCTGCGGCG), described elsewhere [START_REF] Günther | A novel method for efficient amplification of whole hepatitis B virus genomes permits rapid functional analysis and reveals deletion mutants in immunosuppressed patients[END_REF][START_REF] Marlet | Revisiting HBV resistance to entecavir with a phenotypic approach[END_REF]. These PCRs were performed with 300 nM of each primer, 2.5 µL of DNA extract and 12.5 µL of Platinum™ PCR SuperFi PCR Master Mix 2X (Thermo Fisher Scientific, Massachusetts, USA). Cycling reactions were performed on a T3000 (Biometra) with the following cycle parameters: 2 min at 94°C, followed by 40 cycles of 40 s at 94°C, 90 s at 60°C, 90 s at 68°C, with an increment of 12 s after each cycle and a final extension step of 10 min at 68°C. We fused these two overlapping regions of the HBV genome together by PCR, using 1 µL of each diluted PCR product (equimolar) obtained in the first step and 12.5 µL of Platinum™ SuperFi PCR Master Mix 2X. Fusion PCR was performed on the T3000 with the following cycle parameters: 30 s at 98°C, followed by 10 cycles of 10 s at 98°C, 45 s at 72°C and 2 min at 72°C. The whole genome of HBV was amplified by PCR with 300 nM primers P1 and P2, and 25 µL of fusion PCR product. Cycling reactions were performed on a T3000 Biometra with the following cycle parameters: 2 min at 94°C, followed by 40 cycles of 40 s at 94°C, 90 s at 60°C, 180 s at 68°C, with an increment of 12 s after each cycle and a final extension step of 10 min at 68°C.

Next-generation sequencing was performed on the Miseq platform (Illumina, San Diego, USA), with a previously described workflow [START_REF] Guinoiseau | Deep sequencing is an appropriate tool for the selection of unique Hepatitis C virus (HCV) variants after single genomic amplification[END_REF]. Briefly, the sequencing library was prepared with the Nextera XT DNA sample preparation kit (Illumina, San Diego, USA), in accordance with the manufacturer's protocol. Paired-end sequencing reads of 150 base pairs were obtained with the Miseq (Illumina, San Diego, USA) platform. Illumina sequencer output files matching of up to 151-base pair sequencing reads were processed with the Galaxy platform after filtering and checking read quality (FastQC algorithm). Trinity software was used for de novo assembly. The constructed sequences were checked with the Blast option from the National Center for Biotechnology Information (NCBI). Reads were mapped with the Bowtie2 tool onto the best sequence given by the de novo assembly sequences. Nucleotide analysis was performed position-by-position using the mpileup program. Positions with a sequencing depth of over 100X were retained for further analysis. Only variants representing 10% or more of the HBV quasispecies were considered for analysis. For each patient, consensus sequences of the whole HBV genome were compared between samples collected before treatment and at treatment failure. We searched for known HBs Ag escape mutations [START_REF] Colagrossi | Immune-escape mutations and stop-codons in HBsAg develop in a large proportion of patients with chronic HBV infection exposed to anti-HBV drugs in Europe[END_REF][START_REF] Coppola | Clinical significance of hepatitis B surface antigen mutants[END_REF][START_REF] Lazarevic | Clinical implications of hepatitis B virus mutations: Recent advances[END_REF][START_REF] Weber | Genetic variability of the S gene of hepatitis B virus: clinical and diagnostic impact[END_REF], core promoter and pre-core mutations [START_REF] Parekh | Genome Replication, Virion Secretion, and e Antigen Expression of Naturally Occurring Hepatitis B Virus Core Promoter Mutants[END_REF] in these sequences, after alignment with other sequences from the same genotype and reference sequences: HBV/E (AY738145), HBV/D (AF121240) and HBV/I (AB231908) extracted from HBV database (HBVdb) [START_REF] Hayer | HBVdb: a knowledge database for Hepatitis B Virus[END_REF]. HBV strain X02763 from HBVdb [START_REF] Hayer | HBVdb: a knowledge database for Hepatitis B Virus[END_REF], with the usual numbering, was used as the reference sequence for the annotation of mutations. Mutations were identified on the basis of their position in the pre-S1, HBe, HBx and polymerase proteins or their position in the HBs Ag (s) or reverse transcriptase domain (rt), if relevant. Core promoter mutations (CPM) were annotated with their nucleic acid position in the X02763 HBV genome.

Phenotypic drug resistance testing

We had 1.1 units of the HBV genome (pCI_HBV1.1x), corresponding to a consensus sequence identified by NGS on isolates collected before treatment, synthesized in vitro (by GenScript Biotech, the Netherlands) and inserted between MluI and SalI restriction sites of a pCI vector (Promega, Toulon, France), downstream from a CMV promoter. This construct allowed HBV replication after transfection in HuH7 cells. Relevant mutations selected during entecavir treatment were introduced into these clones by site-directed mutagenesis (GenScript Biotech).

Phenotypic resistance assays were performed as previously described [START_REF] Marlet | Revisiting HBV resistance to entecavir with a phenotypic approach[END_REF].

Briefly, Huh7 cells were transfected with pCI_HBV1.1x. Entecavir monohydrate (Merck, Missouri, USA) was added to the cells at six different final concentrations, ranging from 2.5 to 50 000 nM. After four days of culture, intracapsid viral DNA was quantified by duplex realtime PCR. We determined 50% inhibitory concentrations (IC50) by least squares regression in GraphPad Prism v5.0 (GraphPad Software, California, USA). The fold-change in resistance corresponded to the IC50 of entecavir for a given isolate, relative to the IC50 of entecavir for the wild-type HBV genotype D ayw3 strain (pCI_HBVpg1820), which was tested in parallel.

Entecavir IC50 and the fold-change in resistance were determined in three independent experiments and compared in Mann-Whitney tests. Replication capacity relative to the same wild-type strain as before was determined by quantifying HBV DNA levels in six different wells after four days of culture without antiviral drugs. Results are represented as the means and standard errors.

Data availability

All sequences were deposited in the GenBank database (Accession numbers: SAMN16263252-SAMN16263257 for MiSeq data and MW082636-MW082641 for consensus sequences).

RESULTS

Patients

During the study period, HBV genotypic drug resistance testing was performed on 110 clinical isolates. We identified seven isolates associated with entecavir treatment failure but with no detectable Pol/RT resistance mutations. Four isolates were not included because of poor patient compliance with treatment (n=3) or because viral DNA load was too low for amplification by PCR (n=1, 1.3 log IU/mL). The three remaining isolates (P1, P2 and P3) were associated with a partial virological response (PVR) to entecavir in immunocompromised patients (Figure 1).

Patient #1 underwent liver transplantation in the context of decompensated alcohol-related cirrhosis. He was negative for HBs Ag, HBs Ab and HBc Ab before transplantation, but he received a graft from a donor with positive HBc Ab. He received one dose of anti-HBs immunoglobulins (IVHEBEX, 10 000 IU, LFB Biomédicaments) during the anhepatic phase, to prevent HBV infection from the graft. Despite this prophylaxis, he experienced HBV infection leading to acute hepatitis one year later, whilst on mycophenolate mofetil (MMF) and tacrolimus treatment.

Patient #2 underwent kidney transplantation and was treated with MMF, sirolimus and prednisolone. She was anti-HBc Ab-positive and HBs Ag-negative before transplantation. She was diagnosed with asymptomatic HBV infection six years later (she had tested negative for HBs Ag the year before).

Patient #3 had a history of resolved HBV infection (HBc Ab-positive, HBs Ag-negative) before he was diagnosed with chronic lymphocytic leukemia (CLL). He was treated with one dose of rituximab, and with cyclophosphamide and fludarabine for two years before remission of CLL was achieved and treatment stopped. HBV reactivation was diagnosed six years later, in the context of a relapse of CLL with cytolysis. Rituximab is known to be associated with a high risk of HBV reactivation and could have triggered HBV reactivation in this patient [START_REF] Loomba | Hepatitis B Reactivation Associated With Immune Suppressive and Biological Modifier Therapies: Current Concepts, Management Strategies, and Future Directions[END_REF]. Interestingly, this patient remained negative for HBc Ab throughout followup, probably due to the strong B-cell depletion induced by rituximab [START_REF] Feeney | Reactivation of occult hepatitis B virus infection following cytotoxic lymphoma therapy in an anti-HBc negative patient[END_REF].

Patients #1, #2 and #3 reported good treatment compliance, which was confirmed by therapeutic drug monitoring (entecavir plasma concentrations = 0.45, 0.78 and 0.45 ng/mL, respectively). No relevant drug interactions between HBV treatment and concomitant medication were detected. Retrospective genotypic drug resistance testing identified the same Pol/RT sequences in pretreatment (P1_D0, P2_D0 and P3_D0) and post-treatment (P1, P2 and P3) isolates (GenBank MW082636-MW082641). These findings suggest that the PVR to entecavir in these three patients was not associated with the selection of resistance mutations in the Pol/RT domain, or with poor patient compliance. a HBV real-time assay (Abbott); b Architect HBs Ag qualitative, HBs Ab and HBe Ag assays (Abbott); c This rare genotype originates from a recombination between genotypes A, C and G, and circulates in Asian countries, including Laos, the patient's home country [START_REF] Hannoun | An aberrant genotype revealed in recombinant hepatitis B virus strains from Vietnam The GenBank accession numbers of the sequences reported in this paper are AF241407-AF241411[END_REF][START_REF] Huy | New Complex Recombinant Genotype of Hepatitis B Virus Identified in Vietnam[END_REF]. The subtype of this isolate remained undetermined, because aa s127S was not included in the current classification [START_REF] Kay | Hepatitis B virus genetic variability and evolution[END_REF][START_REF] Purdy | A New Algorithm for Deduction of Hepatitis B Surface Antigen Subtype Determinants from the Amino Acid Sequence[END_REF].

Mutations detected before entecavir treatment

Clinically relevant mutations (HBs immune escape or core promoter mutations, CPM) were detected in the consensus sequence of the three HBV isolates collected before entecavir treatment (Figure 2).

Isolate P1_D0 harbored core promoter mutations (CPM) T1753G, A1762T and G1764A (Figure 2).

The last two of these mutations are known to be associated with poor HBe Ag secretion and an increased HBV replication capacity, with no impact on susceptibility to entecavir [START_REF] Leng | Biological characteristics of the A1762T/G1764A mutant strain of hepatitis B virus in vivo[END_REF][START_REF] Parekh | Genome Replication, Virion Secretion, and e Antigen Expression of Naturally Occurring Hepatitis B Virus Core Promoter Mutants[END_REF]. Interestingly, this unique combination of three CPMs has been described once before, in a patient experiencing HBV reactivation following liver transplantation [START_REF] Kim | Evolution of hepatitis B virus sequence from a liver transplant recipient with rapid breakthrough despite hepatitis B immune globulin prophylaxis and lamivudine therapy[END_REF].

Mutation T1753G has not been previously characterized, but mutation T1753C increased the level of replication associated with mutations A1762T and G1764A [START_REF] Parekh | Genome Replication, Virion Secretion, and e Antigen Expression of Naturally Occurring Hepatitis B Virus Core Promoter Mutants[END_REF].

Isolates P2_D0 and P3_D0 harbored HBs Ag immune escape mutations, which are frequently detected in this context of HBV reactivation [START_REF] Salpini | Hepatitis B surface antigen genetic elements critical for immune escape correlate with hepatitis B virus reactivation upon immunosuppression[END_REF]. Isolate P2_D0 harbored a unique set of mutations in the "a" determinant of the HBs Ag: sK122R, sT126I, sP127S and sG145R. The sK122R, sT126I and sG145R mutations have been associated with immune escape and the restoration of viral fitness in lamivudine-resistant isolates (Amini-Bavil-Olyaee et al., 2010; [START_REF] Bock | Selection of hepatitis B virus polymerase mutants with enhanced replication by lamivudine treatment after liver transplantation[END_REF].

The sP127S mutation has been observed only once before, in an isolate from Laos (Olinger et al., n.d.) but another mutation at this position (sP127L) has been described as an immune escape mutation [START_REF] Colagrossi | Immune-escape mutations and stop-codons in HBsAg develop in a large proportion of patients with chronic HBV infection exposed to anti-HBV drugs in Europe[END_REF]. This suggests that the sP127S mutation may also be an immune escape mutation. Isolate P3_D0 harbored the sM133I mutation of the "a" determinant of the HBs Ag MHR region. This mutation has been associated with HBV reactivation [START_REF] Pei | Spontaneous reactivation of hepatitis B virus replication in an HIV coinfected patient with isolated anti-Hepatitis B core antibodies[END_REF], mother-to-child HBV transmission despite vaccination [START_REF] Latthaphasavang | Perinatal hepatitis B virus transmission in Lao PDR: A prospective cohort study[END_REF] and major antigenic changes in the HBs Ag [START_REF] Ie | Prediction of conformational changes by single mutation in the hepatitis B virus surface antigen (HBsAg) identified in HBsAg-negative blood donors[END_REF].

Mutations selected during entecavir treatment

We analyzed the evolution of HBV quasispecies by studying the whole HBV genome by NGS and comparing isolates collected before (P1_D0, P2_D0 and P3_D0) and after (P1, P2 and P3) entecavir treatment failure. Twenty-five non-synonymous mutations had been selected in isolates P1 (n=9), P2 (n=2) and P3 (n=14) (Figure 3). Two of these mutations, both in isolate P1, were considered to be major variants. Their proportion in the HBV quasispecies increased from 0-6 % before treatment to 41-57 % during treatment (Figure 3). The first mutation resulted in simultaneous changes in the spacer domain of the polymerase (A300E) and in the pre-S1 protein (Q120K) (Figure 3). The latter mutation was localized at the N-terminus of pre-S2

(Q2K) in a T-cell epitope [START_REF] Barnaba | Recognition of hepatitis B virus envelope proteins by liver-infiltrating T lymphocytes in chronic HBV infection[END_REF][START_REF] Pajot | Identification of novel HLA-DR1-restricted epitopes from the hepatitis B virus envelope protein in mice expressing HLA-DR1 and vaccinated human subjects[END_REF]. This mutation has been observed in 1.2% of HBV/E isolates [START_REF] Abdou Chekaraou | A novel hepatitis B virus (HBV) subgenotype D (D8) strain, resulting from recombination between genotypes D and E, is circulating in Niger along with HBV/E strains[END_REF][START_REF] Candotti | Molecular characterization of hepatitis B virus in blood donors from Burkina Faso: Prevalence of quasi-subgenotype A3, genotype E, and mixed infections[END_REF][START_REF] Mulders | Low genetic diversity despite hyperendemicity of hepatitis B virus genotype E throughout West Africa[END_REF]. The second mutation, in the pre-core (Q206K) and core (Q177K) proteins, was located at the C-terminal end of the core protein (Figure 3), in a highly conserved protamine-like DNA-binding domain crucial for HBV pgRNA encapsidation and DNA replication [START_REF] Jung | Cterminal substitution of HBV core proteins with those from DHBV reveals that arginine-rich 167RRRSQSPRR175 domain is critical for HBV replication[END_REF]. Interestingly, this is the first description of a mutation at this position. These two mutations were not known to be associated with resistance to entecavir.

Phenotypic assays were performed on recombinant viruses harboring one or both of these mutations, to determine whether they had any impact on the level of resistance to entecavir.

The other 23 mutations were selected during entecavir treatment, but only as minor variants (frequency <40% at treatment failure), suggesting that they were probably not required for a PVR to entecavir. Eight of these mutations were located in the sequence corresponding to the Pol/RT domain, all as minor variants (<25%). None was detected with Sanger sequencing, coherent with their distribution as minor variants. Three mutations were of clinical interest. The first was pre-core mutation W28*, selected in patient #1. The other two were basal core promoter mutations A1762T and G1764A, selected in patient #3 (Figure 3). These precore/basal core promoter mutations are known to be associated with increases in viral replication and decreases in HBe Ag expression [START_REF] Parekh | Genome Replication, Virion Secretion, and e Antigen Expression of Naturally Occurring Hepatitis B Virus Core Promoter Mutants[END_REF]. However, as minor variants, they were considered unlikely to account for entecavir treatment failure and were not further characterized. Mutations rarely (<3%) found in other isolates of the same genotype are represented with a triangle, whereas more frequent mutations are represented with a diamond. Mutations associated with a change in two ORFs are indicated with a dashed line between the two ORFs.

Shades of gray correspond to the pre-C, Core, pre-S1, pre-S2, S and X genes (upper row) and the terminal protein, spacer, polymerase/reverse transcriptase and RNase domains of the polymerase gene (lower row). MHR, major hydrophilic region. HBV strain X02763, with the usual numbering, was used as the reference sequence for annotation.

Phenotypic characterization of mutations selected during entecavir treatment

Recombinant viruses harboring the whole genome of isolate P1_D0, with or without the two relevant mutations selected during entecavir treatment in patient #1, were obtained by in vitro synthesis and site-directed mutagenesis, followed by transfection of the Huh7 cell line. The level of resistance to entecavir and the replication capacity of these recombinant viruses are shown in Figure 4. Variants harboring one or both of these mutations were as susceptible to entecavir and had a similar replicative capacity to the pretreatment isolate P1_D0 (Figure 4).

This suggests that these mutations, alone or in combination, played no direct role in entecavir treatment failure.

DISCUSSION

In a series of 110 patients tested for HBV drug resistance, we identified three patients experiencing entecavir treatment failure despite having good treatment compliance and no detectable Pol/RT resistance mutations. These three patients experienced a PVR to entecavir in a context of immunosuppression.

Previous studies demonstrated that PVR to entecavir does not require selection of resistance mutations in the Pol/RT domain but is associated with HBe Ag positivity, low levels of ALAT and a high HBV DNA viral load at baseline [START_REF] Chen | Antiviral effect of entecavir in nucleos(t)ide analogue-naïve and nucleos(t)ide analogue-experienced chronic hepatitis B patients without virological response at week 24 or 48 of therapy[END_REF][START_REF] Lee | Prolonged Entecavir Therapy Is Not Effective for HBeAg Seroconversion in Treatment-Naive Chronic Hepatitis B Patients with a Partial Virological Response[END_REF][START_REF] Li | Partial virological response to entecavir treatment in nucleos(t)ide-naïve patients with HBeAg-positive chronic hepatitis B is not caused by reduced sensitivity[END_REF][START_REF] Zoutendijk | Entecavir treatment for chronic hepatitis B: adaptation is not needed for the majority of naïve patients with a partial virological response[END_REF]. Despite these findings, most cases of PVR to entecavir remain unexplained. We hypothesized that mutations selected outside of the Pol/RT domain could contribute to PVR to entecavir. Mutations Q206K (pre-core/core), Q120K (pre-S1/pre-S2, in a 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. No mutations were selected in the whole HBV genome of patients #2 and #3. This study provided a unique insight into the evolution of HBV quasispecies during entecavir treatment, addressing the whole HBV genome and not only the Pol/RT domain. We demonstrated that PVR to entecavir does not require selection of any resistance mutation in the whole HBV genome. Our results demonstrate that mutations can be selected outside of the Pol/RT domain during entecavir treatment. These mutations could contribute to entecavir treatment failure by other mechanisms than an increased level of resistance.

Mutations of unknown significance can be selected in the whole HBV genome during entecavir treatment. In this study, mutation Q120K in the pre-S1/pre-S2 protein was selected during entecavir treatment in patient #1. This mutation was localized in a T-cell epitope (pre-S2 1-15) [START_REF] Barnaba | Recognition of hepatitis B virus envelope proteins by liver-infiltrating T lymphocytes in chronic HBV infection[END_REF][START_REF] Pajot | Identification of novel HLA-DR1-restricted epitopes from the hepatitis B virus envelope protein in mice expressing HLA-DR1 and vaccinated human subjects[END_REF] and in a domain required for viral secretion (pre-S2 1-5) (Le [START_REF] Seyec | Role of the Pre-S2 Domain of the Large Envelope Protein in Hepatitis B Virus Assembly and Infectivity[END_REF]. Mutations and deletions at this position tend to be selected in asymptomatic carriers and are associated with reduced secretion of HBs Ag in vitro and in vivo [START_REF] Fan | Prevalence and significance of hepatitis B virus (HBV) pre-S mutants in serum and liver at different replicative stages of chronic HBV infection[END_REF][START_REF] Taghiabadi | Comparison of pre-S1/S2 variations of hepatitis B virus between asymptomatic carriers and cirrhotic/hepatocellular carcinoma-affected individuals[END_REF][START_REF] Wang | Sequence analysis of the Pre-S gene in chronic asymptomatic HBV carriers with low-level HBsAg[END_REF]. This mutation could have been associated with viral escape from cellular immune responses and could have contributed to the partial virological response observed in patient #1. In contrast, in patients #2 and #3, no mutations were selected in the whole HBV genome during treatment, suggesting that PVR to entecavir did not rely on viral escape or resistance. HBs Ag immune escape mutations were selected before treatment in these two patients, as previously observed in the context of HBV reactivation [START_REF] Corti | Viral envelope-specific antibodies in chronic hepatitis B virus infection[END_REF][START_REF] Rehermann | Immunology of hepatitis B virus and hepatitis C virus infection[END_REF].

It is unclear whether treatment with immunosuppressive drugs, such as rituximab, is associated with PVR to entecavir. These drugs have been associated with an increased risk of HBV reactivation [START_REF] Loomba | Hepatitis B Reactivation Associated With Immune Suppressive and Biological Modifier Therapies: Current Concepts, Management Strategies, and Future Directions[END_REF] and delayed response to entecavir treatment in a minority of patients [START_REF] Brost | Entecavir as treatment for reactivation of hepatitis B in immunosuppressed patients[END_REF][START_REF] Yap | Entecavir treatment in kidney transplant recipients infected with hepatitis B[END_REF]. In addition, low ALAT levels at baseline have been associated with an increase in the risk of PVR to entecavir. It has been hypothesized by others that these low ALAT levels were the result of an impaired host immune response, which could increase the risk of PVR to entecavir [START_REF] Lee | Prolonged Entecavir Therapy Is Not Effective for HBeAg Seroconversion in Treatment-Naive Chronic Hepatitis B Patients with a Partial Virological Response[END_REF][START_REF] Li | Partial virological response to entecavir treatment in nucleos(t)ide-naïve patients with HBeAg-positive chronic hepatitis B is not caused by reduced sensitivity[END_REF].

Our findings support this hypothesis and suggest that mutations selected outside of the Pol/RT domain could contribute to entecavir treatment failure, not by an increased level of resistance, but by an immune escape mechanism, in synergy with immunosuppressive drugs.

We developed an original approach to study entecavir treatment failure, based on whole HBV genome analysis, combining deep sequencing and a phenotypic resistance assay. This made it possible to perform a comprehensive evaluation of mutations selected before or during entecavir treatment, potentially associated with treatment failure. The main limitation of this study is the small sample size, which made it impossible to draw reliable conclusions on the association between immune escape mutations and PVR to entecavir.

This study argues for a systematic approach to entecavir treatment failure. In addition to Pol/RT sequencing, therapeutic drug monitoring seems to be useful for detecting poor compliance with treatment or underdosing in some patients. Screening for HBs immune escape mutations may be relevant in immunocompromised patients experiencing HBV reactivation, to confirm the association between these mutations and the PVR to entecavir and to guide treatment adaptation more effectively. By contrast, deep sequencing does not seem more relevant than Sanger sequencing for detecting resistance mutations at treatment failure, as they can be detected as major variants in this context [START_REF] Lee | Pyrosequencing method for sensitive detection of HBV drug resistance mutations[END_REF][START_REF] Lowe | Implementation of Next-Generation Sequencing for Hepatitis B Virus Resistance Testing and Genotyping in a Clinical Microbiology Laboratory[END_REF]. However, in the near future,

HBV treatment strategies will target other steps of the viral cycle and/or aim to modulate the host immune response [START_REF] Cornberg | Guidance for design and endpoints of clinical trials in chronic hepatitis B -Report from the 2019 EASL-AASLD HBV Treatment Endpoints Conference ‡[END_REF][START_REF] Fanning | Therapeutic strategies for hepatitis B virus infection: towards a cure[END_REF]. In this context, deep sequencing and phenotypic characterization of whole HBV genomes from sequential clinical isolates, as performed in this study, will probably be essential, for the identification of viral factors associated with the response to these new antiviral treatments.

Figure 1 :

 1 Figure 1: Biological and clinical follow-up of patients experiencing a partial virological

Figure 2 :

 2 Figure 2: Mutations detected before entecavir treatment

Figure 3 :

 3 Figure 3: HBV mutations selected or repressed during entecavir treatment

Figure 4 :

 4 Figure 4: Phenotypic resistance testing of HBV isolates selected during entecavir

Table 1 :

 1 Clinical and virological profiles of patients before entecavir treatment

	Patient Sex Age	Immunosuppression	HBV	Serotype Metavir	ALAT	HBV	HBs	HBs	HBe
			(years)	Context	Delay before HBV	genotype		score	(IU/L)	DNA a	Ag b	Ab b	Ag b
					infection (years)					(log
										IU/mL)
	#1	M 41	Liver transplant	1	E	ayw4	A2F2	400	7.4	3760 0	924
	#2	F	75	Kidney transplant 6	I c	ayw c	A0F2	26	6.6	2051 5	2094
	#3	M 73	Chronic	17	D	ayw3	F2	57	8.3	320	0	1622
				lymphocytic
				leukemia

ACKNOWLEDGMENTS

We thank the Thellie endowment fund and our generous donors for funding this project. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.


Marion Mortamais
email: marion.mortamais@inserm.fr

PhD Laure-Anne Anne Gutierrez

MSc Kees De Hoogh

PhD Tarik Benmarhnia

PhD Catherine Helmer

Christophe Tzourio

Jean-François Dartigues

Noemie Letellier

Benedicte Jacquemin

PhD Claudine Berr

PhD Jie Chen

Danielle Msc

Vienneau

PhD Isabelle Carrière

PhD Noémie Letellier

PhD Audrey Gabelle

MD Thibault Mura

MD Jordi Sunyer

PhD Bénédicte Jacquemin

Effects of air pollution on cognition

Keywords: cohort, dementia, incidence, elderly, air pollution, fine particulate matter, nitrogen dioxide, black carbon

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION 1

Dementia defines a group of neurological disorders in which deterioration of memory, thinking, 2 behaviour and ability to perform everyday activities leads to disability and dependency. In older 3 adults, the most common form of dementia is Alzheimer's disease (AD, 70% of cases), followed 4 by mixed or vascular dementia (VaD) [START_REF] Lobo | Prevalence of dementia and major subtypes in Europe: A collaborative study of population-based cohorts. Neurologic Diseases in the Elderly Research Group[END_REF]. With nearly 10 million new cases per year [START_REF]Dementia n[END_REF], dementia 5 is a huge burden worldwide. As no effective treatment exists yet, the identification of modifiable 6 risk factors that could be targeted by prevention strategies is an important public health challenge. 7

This is particularly important for AD, in which the neurodegenerative process may begin at least 8 10 years before the onset of clinical symptoms [START_REF] Amieva | Prodromal Alzheimer's disease: successive emergence of the clinical symptoms[END_REF], thus potentially offering a wide temporal 9 window to delay disease onset. 10

Emerging evidence suggests that air pollutants might contribute to the neurodegenerative 11 pathology through oxidative stress, microglia overactivation, and neuroinflammation [START_REF] Block | Air pollution: mechanisms of neuroinflammation and CNS disease[END_REF][START_REF] Calderón-Garcidueñas | Air pollution and brain damage[END_REF]. 12 Moreover, epidemiological studies indicate that ambient air pollution might have a detrimental 13 effect on cognitive function in aging populations [START_REF] Kulick | Longterm exposure to ambient air pollution, APOE-ε4 status, and cognitive decline in a cohort of older adults in northern Manhattan[END_REF][START_REF] Tzivian | Associations of long-term exposure to air pollution and road traffic noise with cognitive function-An analysis of effect measure modification[END_REF][START_REF] Power | Exposure to air pollution as a potential contributor to cognitive function, cognitive decline, brain imaging, and dementia: A systematic review of epidemiologic research[END_REF]. However, only few studies have assessed 14 the link between dementia incidence and air pollutants, such as nitrogen oxides (NOx, NO2) and 15 fine particulate matter (airborne solid and liquid particles smaller than 2.5 micrometres; PM2.5) 16 [START_REF] Grande | Association Between Cardiovascular Disease and Long-term Exposure to Air Pollution With the Risk of Dementia[END_REF][START_REF] Oudin | Traffic-Related Air Pollution and Dementia Incidence in Northern Sweden: A Longitudinal Study[END_REF][START_REF] Cacciottolo | Particulate air pollutants, APOE alleles and their contributions to cognitive impairment in older women and to amyloidogenesis in experimental models[END_REF][START_REF] Chen | Exposure to ambient air pollution and the incidence of dementia: A population-based cohort study[END_REF][START_REF] Jung | Ozone, particulate matter, and newly diagnosed Alzheimer's disease: a population-based cohort study in Taiwan[END_REF][START_REF] Chang | Increased risk of dementia in patients exposed to nitrogen dioxide and carbon monoxide: a populationbased retrospective cohort study[END_REF][START_REF] Carey | Are noise and air pollution related to the incidence of dementia? A cohort study in London, England[END_REF], and even fewer studies have investigated the effects of air pollution exposure on the risk 17 of AD or VaD [START_REF] Grande | Association Between Cardiovascular Disease and Long-term Exposure to Air Pollution With the Risk of Dementia[END_REF][START_REF] Jung | Ozone, particulate matter, and newly diagnosed Alzheimer's disease: a population-based cohort study in Taiwan[END_REF][START_REF] Carey | Are noise and air pollution related to the incidence of dementia? A cohort study in London, England[END_REF]. Moreover, these studies present important methodological limitations. 18 Indeed, most of them relied on population-based health administrative databases for the diagnosis 19 of dementia, a method that is prone to classification bias [START_REF] Chen | Exposure to ambient air pollution and the incidence of dementia: A population-based cohort study[END_REF][START_REF] Jung | Ozone, particulate matter, and newly diagnosed Alzheimer's disease: a population-based cohort study in Taiwan[END_REF][START_REF] Chang | Increased risk of dementia in patients exposed to nitrogen dioxide and carbon monoxide: a populationbased retrospective cohort study[END_REF][START_REF] Carey | Are noise and air pollution related to the incidence of dementia? A cohort study in London, England[END_REF]. The rare longitudinal 20 population-based studies with active search of dementia cases and validated diagnosis concerned 21 only women [START_REF] Cacciottolo | Particulate air pollutants, APOE alleles and their contributions to cognitive impairment in older women and to amyloidogenesis in experimental models[END_REF] and small samples [START_REF] Grande | Association Between Cardiovascular Disease and Long-term Exposure to Air Pollution With the Risk of Dementia[END_REF][START_REF] Oudin | Traffic-Related Air Pollution and Dementia Incidence in Northern Sweden: A Longitudinal Study[END_REF]. 22

As air pollution levels can be modified by public policies with benefits at the population level, it 23 is important to precisely characterize the magnitude of the air pollution effects on dementia. [START_REF] Liu | Multiple Imputation by Fully Conditional Specification for Dealing with Missing Data in a Large Epidemiologic Study[END_REF] this study, we investigated the association between long-term exposure to air pollutants and 25 incidence of dementia in older adults using reliable diagnostic tools in a large population-based 26 cohort in France. 27

METHODS

29

Study population 30

The Three-City Study (3C Study) is a longitudinal, population-based prospective cohort study of 31 community-dwelling older adults [16]. Between March 1999 and March 2001, non-32 institutionalized, ≥65-year-old adults, registered in the electoral rolls of selected districts in 33 Dijon, Bordeaux, and Montpellier (France) were invited to participate through a personal letter 34 (acceptance rate = 37%). In total, 9294 participants were included. The baseline assessment and 35 the following extensive follow-up visits performed every two years included standardized 36 questionnaires, clinical examinations, and detailed cognitive evaluations. 37

Among the 9294 participants originally included, 9251 had a baseline residential address that 38 could be geocoded. Among them, 214 participants with prevalent dementia at baseline and 823 39 participants who did not have any follow-up visit (at 2, 4, 7, 10, and 12 years after baseline) were 40 excluded. Therefore, the present analyses were carried out on 7066 participants for whom 41 complete and validated data on exposure to air pollution, dementia diagnosis, and covariates were 42 available (Figure 1). 43

The study protocol was approved by the Ethics Committee of the Hospital of Kremlin-Bicêtre 44 and Sud-Méditerranée III. A written informed consent was obtained from all participants (consent 45 for research). 46 centre where everybody had a neurological examination at each follow-up visit. Third, an 56 independent committee of neurologists reviewed all potential cases of dementia to confirm the 57

Neurosciences. 64

In this study, all incident cases of all-cause dementia, AD, and vascular/mixed dementia (VaD) 65 during the 12-year follow-up were considered. 66 67

Exposure to air pollution 68

Exposure to PM2.5, black carbon (BC), and NO2 was estimated at the geocoded baseline 69 residential address of each participant using hybrid land use regression (LUR) models [START_REF] De Hoogh | Spatial PM2.5, NO2, O3 and BC models for Western Europe -Evaluation of spatiotemporal stability[END_REF] that 70 Cohorts for Air Pollution Effects (ESCAPE) [START_REF] Eeftens | Spatial variation of PM2.5, PM10, PM2.5 absorbance and PMcoarse concentrations between and within 20 European study areas and the relationship with NO2 -Results of the ESCAPE project[END_REF]. 77

Potential predictor variables of the LUR models included land use characteristics, population 78 density, roads, altitude, distance to the sea, pollutant estimates for 2010 from two long-range 79 chemical transport models (MACC-II ENSEMBLE [START_REF] Inness | The MACC reanalysis: an 8 yr data set of atmospheric composition[END_REF] and the Danish Eulerian Hemispheric 80

Model [START_REF] Brandt | An integrated model study for Europe and North America using the Danish Eulerian Hemispheric Model with focus on intercontinental transport of air pollution[END_REF]), and satellite-derived PM2.5 and NO2 measurements. The final models, described in 81 detail by de Hoogh et al [START_REF] De Hoogh | Spatial PM2.5, NO2, O3 and BC models for Western Europe -Evaluation of spatiotemporal stability[END_REF] explained 72%, 54% and 59% of the spatial variation in the measured concentrations of PM2.5, BC and NO2, respectively. Models were for 100x100m grid cells across 83

Western Europe [START_REF] De Hoogh | Spatial PM2.5, NO2, O3 and BC models for Western Europe -Evaluation of spatiotemporal stability[END_REF]. 84 85 Then, the 2010 model estimates were extrapolated for the 1990-2012 period according to the 86 method used in the ELAPSE study [START_REF] De Hoogh | Spatial PM2.5, NO2, O3 and BC models for Western Europe -Evaluation of spatiotemporal stability[END_REF]. Backward and forward extrapolations were applied at 87 the regional level (i.e. European Classification of Territorial Units for Statistics) to derive the 88 exposures for the other years. This extrapolation was based on the annual mean estimates from 89 the 26 x 26km Danish Eulerian Hemispheric Model, previously downscaled from the original 50 90

x 50km resolution using bilinear interpolation [START_REF] Brandt | An integrated model study for Europe and North America using the Danish Eulerian Hemispheric Model with focus on intercontinental transport of air pollution[END_REF]. 91 92

Individual and contextual covariates at baseline 93

The covariates that could be potential confounders were pre-selected based on the review of 94 literature data. 95 Socio-demographic variables included sex, age, study centre, and education level (primary 96 education: ≤5 years; lower secondary education: up to 9 years; and higher secondary education: 97 >9 years). Health behaviour variables included smoking habits (never/past or current smoker), 98 alcohol intake (none, moderate if <36g per day, or heavy if ≥36g). 99

APOEε4 carrier was defined as the presence of at least one ε4 allele. 100

For each participant, the contextual neighbourhood socioeconomic status was defined using a 101 deprivation index based on the proportion of households without car, of tenants and single parents, 102 unemployment rate, settlement index, and tax household income [START_REF] Letellier | Sexspecific association between neighborhood characteristics and dementia: The Three-City cohort[END_REF] at the IRIS level (Ilots 103 Regroupés pour l'Information Statistique), the finest spatial census unit (2000 residents per unit). 104 105

Statistical analysis 106

The relationships between dementia incidence and PM2.5, NO2, and BC exposure levels were 107 assessed using Cox proportional hazards models that included a marginal intra-municipality correlation, delayed entry, and age as the basic timescale. These models, which used a robust 109 sandwich variance estimator, allow taking into account the correlations between individuals in 110 the same municipality (n=3 in Dijon and Montpellier, and n=6 in Bordeaux). Participants without 111 dementia who died or were lost to follow-up were censored at the last cognitive examination. The 112 date of dementia onset was set as the midpoint between the last follow-up visit without dementia 113 and the first one with dementia. 114

Exposure to air pollution was included as a time-varying variable. For each year of follow-up, the 115 10-year moving average of the past exposure to each pollutant was estimated for each participant. 116

All models were single-pollutant models. 117

First, the crude association between air pollution exposure and risk of dementia (model 0) was 118 investigated. Then, model 1 was adjusted for sex, education level, centre, APOE genotype, 119 deprivation index, alcohol intake, and smoking habit. The proportional hazards assumption was 120 verified by adding the cross-product of each variable with the logarithm of the time variable. 121

In an additional analysis, the potential effect modification (on the multiplicative scale) by sex, 122 centre, education, APOE genotype, and deprivation index was assessed by including an 123 interaction term with air pollutant. 124

The assumption of log-linearity for each pollutant was verified using restricted cubic spline 125 functions with three to five knots. As no strong evidence of departure from linearity for the 126 relation of any of the three pollutants was observed, the results of the proportional-hazards 127 regression analyses were expressed as hazard ratios (HR) with 95% confidence intervals (CI) for 128 every 5µg/m 3 increase in PM2.5 or NO2 levels, and for every 10 -5 /m increase in BC level. 129

Analyses were performed with the SAS software, version 9.4 (SAS Institute). 130 131

Sensitivity analyses 132

Additional analyses were performed after adjusting for household income, family history of 133 dementia (models 1a and 1b), and then for vascular risk factors (diabetes and history of vascular 134 pathology, model 2), considered as mediators in the pathway between air pollution exposure and risk of dementia [START_REF] Ilango | The role of cardiovascular disease in the relationship between air pollution and incident dementia: a population-based cohort study[END_REF]. Model 2 was then adjusted for depressive symptomatology and physical 136 activity (model 3). Analyses were also restricted to non-movers (by excluding participants who 137 moved during the 10 years before baseline). 138

As more than 10% of the entire study population was excluded due to missing data, additional 139 analyses were performed after imputing missing data using the fully conditional specification 140 multiple imputation method [START_REF] Liu | Multiple Imputation by Fully Conditional Specification for Dealing with Missing Data in a Large Epidemiologic Study[END_REF]. Finally, inverse probability weighting (IPW) was implemented 141 to account for the potential attrition bias. 142

The multiple imputation and IPW procedures and the obtained results are described in 143 Table 1 describes the baseline characteristics of the 7066 participants in function of their dementia 152 status. In the whole sample, the median (IQR) age was 73.4 (8.0) years, 62% were women, and 153 37% had more than 9 years of education. The median follow-up duration was 10.0 (6.9) years. 154

Compared with participants without dementia, participants who developed dementia were 155 significantly older, more frequently women, and with lower education level. They were also more 156 likely to live in a deprived neighbourhood. 157

Compared with the study population, participants who were excluded from the initial sample were 158 significantly older and more frequently men (Table S1). 159 160 All baseline home addresses were located in urban areas, as defined by the French National 161

Statistics Institute (INSEE, https://www.insee.fr/en/metadonnees/definition/c1501), and 80% of 162 participants had lived at that address for at least 10 years before enrolment. Figure 2 shows the 163 estimated annual mean concentrations of PM2.5, BC and NO2 for the study participants during 164 the 1990-2012 period. The mean individual exposures estimated at the participants' residential 165 address for the 10 years before dementia onset for participants with incident dementia or before 166 the date of censoring for participants who did not develop dementia were 21.9 (2.6) μg/m 3 for 167 PM2.5, 2.4 (0.3) x10 -5 /m for BC, and 34.2 (7.5) μg/m 3 for NO2 (Table 2). PM2.5 exposure level 168 was modestly correlated with NO2 and BC levels (Pearson's correlations: 0.36 and 0.60). The 169 exposure levels of BC and NO2, two markers of vehicle exhaust, were strongly correlated 170 (Pearson's correlation: 0.80). 171

The associations between air pollutant exposure and incidence of dementia are presented in Table 174 3. PM2.5 exposure level was positively associated with dementia risk in the crude (models 0) and 175 adjusted models (models 1). Adjustment for sex, centre, education, deprivation index, APOE 176 genotype and health behaviours (model 1) substantially decreased the effect size for all-cause 177 dementia and AD. In model 1, a 5µg/m 3 increase in PM2.5 level was associated with an increase 178 by 20% of the risk of all-cause dementia [HR=1.20, 95%CI (1.08-1.32)], by 20% for AD 179 The effect of PM2.5 was not modified by sex, centre, education, APOE genotype, and deprivation 181 index. No relationship was observed between NO2 or BC exposure and risk of dementia (all 182 causes), AD or VaD. 183 Sensitivity analyses showed that adjustment for household income, family dementia history 184 (Table S2) or vascular risk factors (Table S3) did not change the results, but for the association 185 between PM2.5 exposure and VaD risk that was no longer significant (Table S3). 186 Additional adjustments in model 3 (i.e. history of respiratory pathology, depressive 187 symptomatology, and physical activity), exclusion of movers from the study population, and 188 multiple imputation of missing data did not substantially change the results (Tables S3, S4 and 189

S6). 190

The HR values obtained from the IPW analyses were slightly higher than those of the main 191 analyses (HR=1.42, 95%CI [1.30-1.56] per 5µg/m 3 increase in PM2.5 levels for all-cause 192 dementia), but NO2 and BC were again not associated with the risk of dementia (Table S7). 193

This study has several strengths. To date, this is the largest cohort study with repeated clinical 277 evaluations that investigated the effect of air pollution exposure on the risk of dementia. The 278 sample size and the follow-up length provided sufficient power to estimate precisely the 279 magnitude of this effect on the main forms of dementia (i.e. AD and VaD). The dementia 280 aetiological diagnoses were validated by an independent committee, thus limiting the 281 classification bias. 282

The PM2.5 exposure estimates were provided by a fine-scale LUR model with a good predictive 283 power.

CONCLUSION 305

This study provides evidence that long-term exposure to PM2.5 is associated with all-cause 306 dementia, AD, and VaD incidence. By suggesting that exposure to PM2.5 might be a modifiable 307 risk factor of the main forms of dementia in older adults, these results add to the emerging 308 evidences highlighting the urgent need to re-evaluate public policies on PM2.5 emissions. 309 Reducing anthropogenic PM2.5 emissions might have a preventive effect on dementia incidence 310 at the population level. 311 High alcohol intake was defined by an intake >36g per day. a for each year of follow-up, a 10-year moving window of the mean past exposure to each pollutant was estimated for each subject. b for each 5µg/m 3 increase c for each 10 -5 /m increase d after exclusion of all incident cases of dementia other than AD e after exclusion of all incident cases of dementia other than vascular or mixed dementia M0: Cox Proportional Hazards model with delayed entry with age as the basic timescale and birth as the time origin. M1: M0 adjusted for sex, centre, education, APOE genotype, deprivation index, alcohol intake, and smoking habits.

  diagnosis and aetiology, according to the criteria of the Diagnostic and Statistical Manual of 58 Mental Disorders, fourth edition (American Psychiatry Association, 2000). 59 Cases of AD were classified according to the National Institute of Neurological and 60 Communicative Disorders and Stroke-Alzheimer's Disease and Related Disorders Association, 61 and cases of mixed/vascular dementia according to the National Institute of Neurological 62 Disorders and Stroke-Association Internationale pour la Recherche et l'Enseignement en 63

  were developed for Western Europe within the framework of the Effects of Low-Level Air 71 Pollution: A Study in Europe (ELAPSE). Briefly, PM2.5 and NO2 concentration data for the year 72 2010 derived from the European Environment Agency AirBase network that collects data 73 recorded at routine monitoring stations (including traffic, industrial and underground sites). The 74 annual mean BC concentrations (measured as PM2.5 absorbance based on reflectance 75 measurement of the filters) for the 2009-2010 period were derived from the European Study of 76

  Among the 7066 participants at baseline, 791 incident cases of dementia were diagnosed during 147 the 12 years of follow-up (incidence = 1.22 dementia cases per 100 person-years): 541 cases of 148 AD (68%), 155 cases of VaD (20%), and 95 cases of other dementia types. During the study 149 period, 1543 participants died (22%), and 1911 (27%) were lost to follow-up or refused to 150 continue the study. 151

[

  HR=1.20 (1.09-1.32)], and by 33% for VaD [HR=1.33 (1.05 -1.68)]. 180

Figure 1 . 22 Figure 2 .

 1222 Figure 1. Flowchart of the Three-City study with a 12-year follow-up.

Table 1 .

 1 Participants' characteristics at baseline

		All Participants	All-cause dementia cases over the 12-year follow-up
		Total, n=7066 Non-cases, n=6275	Cases, n=791	p-value a
		n (%) or median ( IQR)
	Age at baseline, years	73.4 (8.0)	72. 9 (7.8)	77.1 (7.1)	<0.001
	Duration of follow-up, years	10.0 (6.9)	10.5 (7.1)	6.7 (4.8)	<0.001
	Sex, women	4359 (61.7%)	3842 (61.2%)	517 (65.4%)	0.024
	Study centre
	Bordeaux	1616 (23.0 %)	1338 (21.3%)	278 (35.2%)	<0.001
	Dijon	4034 (57.0 %)	3647 (58.1%)	387 (48.9%)
	Montpellier	1416 (20.0%)	1290 (20.6%)	126 (15.9%)
	Education
	Primary ( ≤ 5 years)	2323 (32.9 %)	1971 (31.4%)	352 (44.5%)	<0.001
	Lower secondary ( 5 -9 years)	2108 (29.8 %)	1925 (30.7%)	183 (23.1%)
	Higher secondary (> 9 years)	2635 (37.3 %)	2379 (37.9%)	256 (32.4%)
	Deprivation index	-0.27 (2.02)	-0.29 (1.99)	-0.16 (2.02)	0.006
	APOE ɛ4 allele carriers	1397 (19.8%)	1185 (18.9%)	212 (26.8%)	<0.001
	Smoking habits
	Never	4374 (61.9%)	3850 (61.3%)	524 (66.3%)	0.008
	Past or current	2692 (38.1%)	2425 (38.7%)	267 (33.7%)
	Alcohol intake
	None	1438 (20.4%)	1257 (20.0%)	181 (22.9%)	0.094
	Moderate	5067 (71.7%)	4510 (71.9%)	557 (70.4%)
	High	561 (7.9%)	508 (8.1%)	53 (6.7%)

a Wilcoxon test for quantitative variables, and Chi2 test for qualitative variables. Abbreviations: APOE, apolipoprotein E; IQR, Interquartile Range. The deprivation index was based on the proportion of households without car, tenants and single parents, unemployment rate, settlement index, and tax household income at the IRIS level (Ilots Regroupés pour l'Information Statistique), the finest spatial census unit (2000 residents per unit). Higher scores indicate more deprived neighbourhood areas (scores ranged from -4.19 to 10.75 in our study).

Table 2 .

 2 Participants' mean exposure levels to air pollutants during the 10 years before the event or censoring.

	Air pollutants	Mean (SD)	Median	Range
	PM2.5 (µg/m3)	21.9 (2.6)	21.3	14.6 -31.3
	BC (10 -5 /m)	2.4 (0.3)	2.3	1.4 -4.6
	NO2 (µg/m3)	34.2 (7.5)	32.8	12.8 -91.8
	Abbreviations: PM, Particulate Matter; BC, Black Carbon; SD, Standard Deviation

Table 3 .

 3 Hazard ratios (95% CI) for the association between air pollutants a and dementia risk during the 12-year period of follow-up

				M0		M1
		Ntotal Ncases	HR (CI 95%)	p	HR (CI 95%)	p
				All-cause dementia
	PM2.5 b	7066	791	1.33 (1.17-1.50) <0.001	1.20 (1.08-1.32) <0.001
	BC c	7066	791	0.91 (0.58-1.42) 0.677	1.10 (0.84-1.43)	0.503
	NO2 b	7066	791	0.96 (0.88-1.04) 0.292	1.01 (0.95-1.08)	0.703
				Alzheimer's disease
	PM2.5 b	6816 d	541	1.33 (1.14 -1.56) <0.001	1.20 (1.09 -1.32) <0.001
	BC c	6816 d	541	0.82 (0.53 -1.27) 0.378	1.00 (0.80 -1.25)	0.991
	NO2 b	6816 d	541	0.94 (0.87 -1.01) 0.076	1.01 (0.96 -1.05)	0.777
				Vascular or mixed dementia
	PM2.5 b	6430 e	155	1.35 (1.03 -1.77) 0.029	1.33 (1.05 -1.68)	0.019
	BC c	6430 e	155	1.29 (0.65 -2.55) 0.462	1.47 (0.80 -2.68)	0.213
	NO2 b	6430 e	155	0.99 (0.87 -1.13) 0.915	1.01 (0.88 -1.17)	0.854

Abbreviations: APOE, apolipoprotein E; BC, Black Carbon; CI, Confidence Interval; HR, Hazard Ratio; PM, Particulate Matter.

ACKNOWLEDGMENTS

We thank Elisabetta Andermarcher for English editing.

FUNDINGS

Marion Mortamais is supported by a post-doctoral fellowship from the Fondation de France (Allocation Postdoctorale n°engagement 00089836). The 3C Study is carried out under a partnership agreement between the Institut National de la Santé et de la Recherche Medicale (INSERM), Victor-Segalen Bordeaux-2 University, and Sanofi-Aventis. The Fondation pour la Recherche Médicale supported the preparation and initiation of the study. The 3C The study was also supported by the Caisse Nationale Maladie des Travailleurs Salaries; Direction Générale de la Santé; MGEN; the Institut de la Longevité; Agence Nationale de la Recherche ANR PNRA 2006 (06-01-01) and Longvie 2007 (LVIE-003-01); Agence Française de Sécurité Sanitaire des Produits de Santé; the Regional Governments of Aquitaine, Bourgogne, and Languedoc-Roussillon; the Fondation de France; the Ministry of Research-INSERM Programme Cohorts and collection of biological material; Fondation Plan Alzheimer" (FCS 2009-2012); the Caisse Nationale de Solidarité pour l'Autonomie (CNSA); Novartis ; and the Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (Anses, grant N° 2019/1/116).

The funders had no role in the design and conduct of the study, collection, management, analysis, and interpretation of the data; and preparation, review, or approval of the manuscript.

DECLARATION OF INTEREST

The authors do not have any conflict of interest to declare.

Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:


S Gazzotti

F Ferlay

L Meunier

P Viudes

K Huc

A Derkazarian

J-P Friconneau

B Peluso

J-P Martins

Virtual and Augmented Reality Use Cases for Fusion Design Engineering

Keywords: Virtual Reality, Augmented Reality, WEST tokamak, ITER, Test Blanket System, Health & Safety, MIFI

. Data preparation process has been optimized, reducing time cost to convert CAD data to an interactive and immersive model. For the WEST Project, VR engineers developed immersive scenes to simulate assembly sequences, to check component introduction using physics and to show 2D temperature data from diagnostics in a 3D environment. For the TBM case, health and safety oriented model environment is developed. Features for team simulated human access and hands on operations for As Low As Reasonably Achievable (ALARA) optimisation process with motion-tracking system and virtual avatars with ventilated protective pressurised suit simulating worker protection in nuclear environment. For MIFI application, a specific assembly tool has been developed for TF coils interface located on the top and at the bottom junction. During a first phase, the development was driven by iterative design loop (CAD modelling analysis and cinematic by using VR simulations). By means of interactive simulations and immersive conditions, the interface connection task was validated against feasibility including accessibility. AR has also been introduced in the process since a physical mock-up has been augmented to consider the constraint environment of TF Coils. For TBM application, on site AR simulation of the port Cell equipment will provide enhanced environment description. Collaborative immersive context, digital twins and combination between AR/VR & physical mock-ups for validation of maintenance since component design phase will constitute challenge for the near future.

Introduction: 10 years of evolution

Complex environment for Fusion projects such as WEST or ITER tokamak pushes the boundaries of physics and makes technical teams find new ways to think.

In the last decade, CEA IRFM has integrated the use of Virtual Reality in the design process following market technology evolution. In 2017, CEA was able to use VR technologies for immersive studies however, limits were reached such as efficient conversion process from CAD model or limited physical interactions when trying to study specific assembly operations. [START_REF] Meunier | Virtual Reality: Lessons Learned from WEST Design and Perspectives for Nuclear Environment[END_REF] Today, boundaries are pushed further using the most advanced XR (eXtended Reality) technologies:

WEST tokamak

The Tore Supra tokamak has been transformed in an Xpoint divertor fusion device within the frame of the WEST (W-for tungsten- Being able to visualise and manipulate virtual elements at true scale holds incredible possibilities into solving problems and is a complete asset into the design process.

With new specific tools such as PIXYZ software, the time process from CAD to VR scene has been drastically reduced using scripting and library data. We can note great improvements in graphic rendering, frame rates and tracking quality on VR devices with the constant evolution of technology.

Although Virtual Reality was already used for static analysis such as design review and clash detection, today expectations can be higher with real time simulations.

Using light projections in Unity software, we are able to add infrared diagnostic 2D temperature data in a 3D environment allowing technicians to identify hotspots precisely.

With the "Assembly" module of INTERACT plugin in unity software, environments allows to build real assembly scenarios and check feasibility. In a near future, artificial intelligence and interconnected IOT will take into account parameters such as emotional state or connected tools data in order to help operators completing the whole assembly sequence.

Recently, human access with Augmented Reality is becoming part of the design process. Microsoft Hololens 2 makes possible to stack virtual elements over a real context allowing accessibility studies and design review directly on site. With a 52° field of view and improved technical characteristics this embedded technology has made a significant progress and becomes interesting for this type of analysis. Further evolution will make AR more accessible with enhanced computations and stronger interaction capabilities.

MIFI : Magnet Infrastructure Facilities for ITER

Assembling of ITER components is a major challenge considering the large size and weight of components and the high accuracy that has to be reached. As part of the Magnets Infrastructures Facility for ITER (MIFI) agreement between CEA and ITER Organisation, CEA works on the assembly sequence of ITER TF coils OIS (Outer Interface Structure) composed of shear pins and bolts located in a small space which considerably complicates their manipulation and assembling.

A specific tool developed by the CEA, and inspired by tele-manipulators, uses complex kinematics and a counterweight in order to be balanced in any position, allowing the operator to easily manipulate the 35 kg bolt.

As the assembly sequence and the environment are very constrained, accessibility has been checked thanks to Virtual Reality through immersive scene using contact interaction and force feedback with haptic robotic arms and XDE interactive physics engine developed by CEA-LIST. The operator is able to immerse himself under the coil and manipulate the counterweight trying to fit the pin in his hole with high tolerances. Additional data such as augmented screen streaming camera video in real time are added in order to have a better view of the context.

The handling and positioning tool was also successfully demonstrated on a physical mock-up of the lower part of the coil structure at MIFI [START_REF]ITER NEWSLINE -Assembly Zero[END_REF] The operator is placed in situation and can manipulate the tool equipped with the counterweight.

AR simulation is added to superimpose the virtual workspace using a Microsoft Hololens 2 device allowing him to apprehend the With iterative studies between design, analysis and simulation using VR/AR we were able to reduce the time to market to design such a complex tool.

TBM : Test Blanket Module System

For ITER Organisation concerning the development of the Test Blanket Modules replacement operations in port cells, CEA develops design recommendation by combination of Virtual Reality Tools and Nuclear Engineering Design activities in Support of Test Blanket Modules Systems development.

To implement ALARA optimisation process since early engineering phase of TBM component, immersive and interactive technologies allows to find ways to optimise component design to minimise occupational exposures of worker, implementing therefore recommendations from International Atomic Energy Agency [START_REF]Optimisation of Radiation Protection in the Control of Occupational Exposure IAEA[END_REF].

Several scenario and immersive scenes were developed.

During TBM design process, working sessions on VR setup have been organised with ITER, nuclear facility operators, integrators and component designers.

Using virtual avatar equipped with nuclear bubble suit (see fig 9) and advanced operator body tracking, enable to maximise the simulation realism and enhance validation step of TMB tasks by VR.

In view of operator working conditions assessment, we plan to support ergonomics studies thanks to motion capture and force-feedback systems allowing real-time information during design review and take into account Human factor and associated ergonomics requirement.

Cutting & Welded tools were implemented in the virtual environment with model of interaction and visual feedback such as process effect like production of metal chips.

The use of mixed reality capture in order to record a real user interacting with a virtual world is an asset allowing real time viewing of a VR experience without the use of an avatar.

CEA team also prepared an Augmented Reality simulation in order to study on the workers accessibility. The model is displayed through wearable glasses (Hololens 2) with CEA embedded software features that allows to superimpose real and virtual environment.

This set up has been experimented in situ (IO site, inside the TBM Equatorial Port Cell #16 before commissioning), in order to compare the virtual model to the real environment enhance immersion conditions. [3] Working sessions allowed confirming the benefits of Augmented Reality in situ, in particular for combined assessment of Design Integration and Health and Safety teams. Human accessibility studies are facilitated by a direct immersion with the real context enhanced with the 3D hologram of the model.

The accuracy of the virtual/real superimposition is relevant for global checking between models and as built however, enhancement of performance can be expected by proper calibration process and additional features.

Conclusion: Next steps and Perspectives

Following a first thesis on operators, accessibility studies for maintenance scenario [START_REF] Louison | Operators' accessibility studies for assembly and maintenance scenarios using virtual reality[END_REF] The views and opinions expressed herein do not necessarily reflect those of the ITER Organization.

Fig. 1 .

 1 Fig.1. CEA IRFM VR Room -configuration

 6

 6 DoF Virtual Reality headsets such as Oculus Rift S and Vive Pro Eye  Augmented Reality (AR) with Microsoft hololens 2 (2019)  Virtual manikin with motion capture using ART suit (24 points) and LS Suit with Vive tracker technology (8 points)  Haption haptic arms with force feedback  High Speed Computer with RTX graphic cards  Real time hand tracking (Ultraleap Motion)  Unity software with Interact Plugin [11] using CEA XDE interactive physics engine developed by CEA-LIST  Pixyz software optimising data process from CAD to FBX 3D models [10]

Fig. 2 .

 2 Fig.2. CEA IRFM VR Room -Hardwares & Softwares

Fig. 4 .Fig. 5 .

 45 Fig.4. infrared data 3Dviewing

  future work will focus on qualification of immersive simulation for feasibility studies of human operators. Include Dose rate elements in AR and VR studies as part of the ALARA process  Investigate and evaluate Advanced calibration methods considering specific features  Investigate XR multi users for full intervention scenario (e.g. injured people evacuation)  Integrate additional model interaction feed back like sounds in the real time simulation  Develop a TBM Pipes Forest Digital Twin by mechanical behaviour model into VR to simulate displacement based on pipe stress analysis results for replacement sequence assessement and validation  Virtual commissioning : Augmented/Mixed Reality on physical TBM Pipe Forest Mock-Up  Coupling physical ergonomic work-bench & AR

	Several areas of investigation and development are
	already identified:
	 Simulation of the TBM representative
	replacement task with an operator in bubble suit
	and umbilical onto a mixed AR & Physical
	mock-up.
	 Perform	design	review	and	plan
	assembly/reconfiguration sequences in situ in
	order to gain confidence in the feasibility
	 develop adequate user interface for interacting
	between real and virtual environment
	 6 Disclaimer