Immune priviledged sites and HSV resistance: experience of the Regavir platform. Ophtalmologica Brussels November 27th, 2014
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1 Immune priviledged sites and HSV resistance: experience of the Regavir platform. Ophtalmologica Brussels November 27th, 2014
2 A Reference and Service Center created in 2009 by the funding received from the Belgian National Cancer Plan (Federal Public Service PUBLIC HEALTH, FOOD CHAIN SAFETY and ENVIRONMENT Our aim: the diagnosis and typing of drug-resistant herpesviruses in immunocompromised patients that fail antiviral therapy and improvement of the scientific background
3 Number of samples per hospital analyzed by RegaVir (January November 2014) Country City Hospital Number of samples Aalst OLV Ziekenhuis Aalst-Asse-Ninove 15 ZNA Middelheim 27 Antwerpen ZNA Jan Palfijn 1 ZNA Stuivenberg 4 UZ Antwerpen 27 Bonheiden Imeldaziekenhuis 5 Brugge AZ Sint Jan Brugge-Oostende 50 Belgium Brugmann Ziekenhuis - Brussel 5 Hôpital Erasme 61 Brussels CHU Saint Luc 14 CHU Saint Pierre 167 UZ Brussel 15 CHIREC 4 Charleroi CHU Charleroi 2 Ghent UZ Gent 49
4 Number of samples per hospital analyzed by RegaVir (January November 2014) Country City Hospital Number of samples Hasselt Jessa Ziekenhuis 23 Kortrijk AZ Groeninge Korthrijk 2 Leuven UZ Leuven 136 Belgium Liège CHU de Liège - Sart Tilman 33 CHR de la Citadelle 21 Roeselare-Menen H.-Hartziekenhuis Roeselare-Menen 1 Sint-Truiden St Trudo Ziekenhuis 2 Yvoir CHU Mont-Godinne
5 Number of samples per hospital analyzed by RegaVir (January November 2014) Country City Hospital Number of samples Paris Hôpiral de la Salpêtrière 6 Hôpital Necker-Enfants malades 3 France Luxembourg The Nederlands USA Lille CHU Lille 2 Lyon Centre de Biologie et Pathologie Est, Hospices Civils de Lyon 30 Suresnes Hôpital Foch 1 Luxembourg Amsterdam Benioff Children's Hospital, San Francisco Centre Hôspitalier de Luxembourg 9 Centre Hôspitalier de Kirchberg 1 Academisch Medisch Centrum Amsterdam 10 West Virginia West Virginia University 3 2 Total number of samples encoded: 675 (national) + 67 (international) = 742 Some of the samples analyzed for more than one virus! Total number of analysis:
6 Number of tests per virus performed by RegaVir (January November 2014) Number of tests HSV-1 HSV-2 VZV HCMV HHV-6 EBV KSHV Parainfluenza virus Adenovirus Polyomavirus Total number of analysis: 807
7 Types of samples received by RegaVir (January November 2014) HSV-1 HSV-2 VZV HCMV HHV-6 Blood Serum Plasma CSF Eye fluid Urine Swab from lesion (mouth) - Biopsy Mouth rinse Respiratory sample Vaginal fluid Bone marrow Not available Total
8 Types of samples analyzed by RegaVir received by RegaVir (January November 2014) EBV HHV-8 (KSHV) Parainfluenza virus Adenovirus Polyomavirus Blood Serum Plasma CSF Urine Biopsy Bone marrow BAL Virus isolate Total
9 Number of patients for whom 1, 2, 3, or >3 samples were analyzed 300 Number of patients >3 Samples analyzed per patient Virus compartmentalization Evolution of viral population
10 Type of analysis performed by RegaVir Virus Phenotyping Genotyping Phenotyping Genotyping Negative sample* Total number of tests performed HSV HSV VZV HCMV HHV EBV 13** KSHV 3** 9 12 Adenovirus BK Polyomavirus Parainfluenza virus * A sample was considered negative when it was not amplifiable by polymerase chain reaction (PCR) and/or when the virus could not be isolated in cell culture. ** No genotyping was performed; only viral detection was carried out. 807
11 Proportion of HSV-1 samples showing drug-resistance as determined phenotypically and/or genotypically HSV-1 drug-resistant viruses: 57.4% (70/122) Number of samples Negative wild-type TK mutants DNA pol mutants Double mutants
12 Proportion of HSV-2 samples showing drug-resistance as determined phenotypically and/or genotypically HSV-2 drug-resistant viruses: 72.3% (68/94) Number of samples Negative wild-type TK mutants DNA pol mutants Double mutants
13 Proportion of VZV samples showing drug-resistance as determined phenotypically and/or genotypically 50 VZV drug-resistant viruses: 25.5% (14/55) Number of samples Negative wild-type TK mutants DNA pol mutants Double mutants
14 Proportion of CMV samples showing drug-resistance as determined genotypically CMV drug-resistant viruses: 27% (90/334) Number of samples Negative wild-type 65 Pul97 mutants DNA pol mutants Double mutants Unknown significance Two UL97 mutants resistant to maribavir Maribavir + ganciclovir = antagonistim no combination 90
15 Proportion of HHV-6 samples showing drug-resistance as determined phenotypically and/or genotypically 20 HHV-6 drug-resistant viruses: 55.6% (5/9) Number of samples Negative wild-type TK mutants DNA pol mutants Double mutants
16 Immune privileged sites Sites able to tolerate the introduction of antigens without eliciting an inflammatory immune response However, immune priviliged sites are neither isolated nor passive in its interactions with the immune system (complex barriers separate immune-priviliged sites from the circulation) Immune privilige is thought to be an evolutionary adaptation to protect vital structures from the potentially damaging effects of the inflammatory immune response. They include: - CNS: brain + spinal cord - Eye - Placenta and fetus - Testes
17 RegaVir platform: 20 ocular samples (Fluids and swabs) Wild-type Drug-resistance Negative HSV (TK) 1 HSV-2-1 (TK) 1 VZV 3 4 (TK) 1 CMV 1 1 (DNA pol) 1
18 Herpetic keratitis (HSV-1) in a renal transplant patient after 6 months valacyclovir therapy Left eye TK: del. C Nts RV-570 TK: del. C Nts W259stop RV-573 Right eye 10/01/14 15/01/14 RV-574 TK : T245M The patient had ocular VZV ; samples negative for VZV
19 Characterization of HSV-1 isolates recovered from patients suffering from herpetic keratitis (Duan et al, 2008) 173 immunocompetent patients with herpetic keratitis (HK) 11 isolates (6.4%) were ACV r - 10 mutations in the viral TK - 5 isolates were cross-resistant to GCV - 1 isolate was cross-resistant to GCV and PFA
20 RegaVir platform: 78 CSF samples Wild-type Drug-resistance Negative HSV (TK) 0 HSV (4 TK 4 Pol) 7 VZV 7 6 (4 TK 2 Pol) 12 HCMV 13 2 (1 UL97 1 Pol) 7 HHV Adenovirus EBV 0 0 1
21 Characterization of HSV-1 isolates recovered from the CSF of patients suffering from herpetic encephalitis Patient 1 (immunocompetent): 72 years old man with HSV-1 encephlitis under acyclovir therapy. He died under ACV treatment. - CSF DNA isolation sequencing HSV TK G59W mutation Patient 2 (immunocompetent): 45 years old man with HSV-1 encephlitis under acyclovir therapy. His treatment was changed to foscarnet and he survived - CSF DNA isolation sequencing HSV TK G59W mutation
22 Schematic illustration of herpes simplex virus (HSV) type 1 load in cerebrospinal fluid (CSF) throughout the course of disease. FIGURE 1: Schematic illustration of herpes simplex virus (HSV) type 1 load in cerebrospinal fluid (CSF) throughout the course of disease. Symptoms began approximately 4days prior to admission to our hospital. On the day of admission (day 1), triple antimicrobial therapy was started and deescalated to acyclovir monotherapy when the positive HSV polymerase chain reaction result was obtained on day 2. At this point, viral load was 411,000 genome equivalents (geq)/ml, which increased to 1,420,000geq/ml on day 6, at which time foscarnet was added to the therapeutic regime. By day 9, HSV DNA was no longer detectable in CSF. Treatment with acyclovir and foscarnet was continued until day 19. [Color figure can be viewed in the online issue, which is available at ANN NEUROL 2010;67:
23 Evolution of HSV-1 strain in the CSF of a 50-years-old woman suffering from herpetic encephalitis Mutations in HSV-1 TK Adenosine (A) del. in a string of 4As (Nts ) Adenosine (A) del. in a string of 4As (Nts ) Adenosine (A) del. in a string of 4As (Nts ) Adenosine (A) del. in a string of 4As (Nts ) RV-171 RV-172 RV /02/ /03/ /03/2011 Mixed populations of wild-type and drug-resistant virus Mixed populations of wild-type and two different drug-resistant viruses Pure population of Drug-resistant virus
24 HSV-2 Heterogeneity - compartmentalization in a HSCT recipient with a primary infection ACV 15 mg/kg/8 h PFA 6g 2x/day RV-194 RV-195 RV-196 RV-197 RV-200 RV-201 RV-202 RV-203 RV-204 RV-205 RV-206 Phenotyping wt ACV R PFA S ACV r PFA R ACV r PFA R ACV r PFA R ACV r PFA R ACV r PFA R ACV r PFA R ACV r PFA R ACV R PFA R ACV r PFA R 14/04/ /04/ /05/ /06/ /06/201 1 Oral Skin swab Skin swa b Skin swab Right hand Left foot Sole of left foot Left hallux Palm of left hand Left index Oral
25 HSV-2 Heterogeneity compartmentalization emergence of novel mutations in a HSCT recipient with a Mutant primary infection Isolate variants RV-194 Wild-type A606V* RV-196 RV-197 V842M* R964H* *Mutations detected by Sanger sequencing are highlighted.
26 HSV-2 Heterogeneity & compartmentalization in a HSCT recipient primary infection Isolate Mutant variants Number of clones isolated RV-194 Wild-type 5/5 (100%) RV-196 A606V* 0/21 T934A 21/21 (100%) K533E 2/21 (9.5%) RV-197 C625R 5/21 (23.8%) R628C 5/21 (23.8%) S725G 3/21 (14.3%) V842M* R964H* 5/21 (23.8%) *Mutations detected by Sanger sequencing are highlighted.
27 HSV-2 Heterogeneity & compartmentalization in a HSCT recipient primary infection Isolate Mutant variants % mutant variants (deepsequencing) Number of clones isolated RV-194 Wild-type Wild-type 5/5 (100%) A606V* /21 RV-196 Y823C /21 V842M 1.0 0/21 T934A /21 (100%) K533E 3.4 2/21 (9.5%) A606V 6.0 0/21 G617S 1.7 0/21 C625R 8.0 5/21 (23.8%) RV-197 R628C 1.8 5/21 (23.8%) S725G 1.1 3/21 (14.3%) A840T 1.5 0/21 V842M* /21 R964H* /21 (23.8%) *Mutations detected by Sanger sequencing are highlighted.
28 HSV-1 Heterogeneity in an allo-hsct (16/05/14) patient Isolate Mutant variants (Sanger sequencing) % of mutant variants (deep-sequencing) Plaque purified clones RV-683 (02/07/14) RV-709 (09/08/2014) RV-714 (29/08/14) TK: A189V Pol: // TK: A189V Pol: // TK: T183P R222H Pol: L778M TK: A189V (99.7%) G del Nts (1%) Pol: // TK: T183P (4.1%) A189V (95.2%) R222H (4.2%) Pol: L778M (4.7%) TK: T183P (97.9) R222H (99.8) Pol: L778M (99.7%) Clone 1 & clone 2 TK: A189V Pol: // Clone 1 TK: T183P R222H Pol: L778M Clone 2 TK: A189V Pol: // Clone 1 & clone 2 TK: T183P R222H Pol: L778M RV-719 (19/09/14) RV-719 bis (19/09/14) TK: T183P R222H Pol: L778M TK: T183P mixed A189V mixed Pol: L778M mixed L802F mixed TK: T183P (97.1) R222H (95.9) Pol: L778M (98.5%) TK: T183P (33.3%) A189V (62.4%) R222H (4%) Pol: L778M (30.4%) L802F 62.5%) Clone 1 & clone 2 TK: T183P R222H Pol: L778M Clone 1 TK: A189V Pol: L778M Clone 2 TK: T183P Pol: L778M 20/06/14 09/07/14 Zovirax 01/08/14 01/09/14 Foscavir Cidofovir RV-683 RV-709 RV-714 RV-719 RV-719bis
29 Take-home message Herpesvirus infections in the CNS and cornea: Risk of development of drug-resistance in both immunocompetent and immunocompromised patients Compartimentalization Viral heterogenicity New genotypic resistance mutations continue to be identified Multiple drug-resistance strains Infection with different drug-resistant viruses Single mutation confering multiple drug-resistance
30 RegaVir Rega Institute Laboratory Virology and Chemotherapy Sarah Gillemot Anita Camps Steven Carmans Graciela Andrei Robert Snoeck Laboratory Immunobiology Pierre Fiten Ghislain Opdenakker This work was supported by the action 29 of the National Cancer Plan (Federal Public Service Public health, food chain safety, and environment ) for translational research UZ Leuven Laboratory Virology Katrien Lagrou Marc Van Ranst
31 Contact: RegaVir Rega Institute for Medical Research Minderbroedersstraat 10 B-3000, Leuven Belgium Tel: / Fax: Website:
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