Multiplex immunoassay (MIA) based on xMAP© (Luminex) Technology Measles IgG performance indicators RIVM Centre for Infectious Disease Control The Netherlands Rob van Binnendijk Fiona van der Klis Luminex Technology xMAP®

Luminex xMAP technology 100 bead regions

Multiplex immunoassay detection Open-architecture xMAP Technology enables multiplexing of biological tests (assays), reducing time, labor, and costs over traditional methods such as ELISA, western blotting, PCR, and traditional arrays Antigen linked to color coded beads Red laser reads the bead internal dye fluorescence Green laser detects the amount of antibody bound in MFI (median fluorescent intensity)

Application: Immune Surveillance vaccine coverage (IgG seropositives) immunity in the population: correlate of protection (CoP) > Detect gaps in immunity, groups/populations at risk

Multiplex Immuno Assay (MIA) : IgG quantitatively bead + measles ag (purified Edmonston, RIVM grade) IgG in sample detection (anti-IgG RPE) bead + mumps ag (Jeryl Lynn) + rubella ag (HPV-77)

Edmonston virus produced on Vero cells (bioreactor) concentrated by ultra filtration (1000 kD) ultracentrifugation > disc. 20/40/70 sucrose gradient 40/70 virus fraction freeze-dried (1-5 mg/ml)

Measles seroprevalence 2006/07 (Dutch cohort) Mollema et al, Epidemiol and infect 2013

MIA > correlation with virus neutralizing antibodies ? Plaque-reduction neutralization (PRN) current standard for measuring humoral protection in measles Edmonston (A-type) lab-strain measles virus based on serum dilution resulting in 50% reduction of infectious centers/plaques (titer) WHO 3rd international standard to express titers in IU/ml estimated threshold for clinical protection 120 mIU/ml But: few clinical surveys support the protective threshold PRN is labor intensive, when compared to enzyme immunoassay (EIA) tests and serum/standard much more prone to variation Serum dilution 4log titration 

* * MIA > correlation with glycoprotein-specific assays ? H F Measles F-/H- transfected cell-lines (de Swart et al. 1998) serum antibody titrations staining with FITC-conjugated anti-human IgG FACS fluorescence analysis sensitivity > EIA (Hartter et al. 2000) mel/JuSo H * F * mel/JuSo mel/JuSo

MIA(IgG) correlation with anti-F, anti-H and PRN Cohort: MMR-1 vaccinated children 4-8y (n=96) Presera (n=96) van de kinderen (4-8 jaar) van scholen uit de Biblebelt (2013); 1x MMR gevaccineerd, vóór expositie aan mazelen (1e afname). 1. Correlatie IgG(MIA) tegen anti-H (test van Rik, = FACS-Elisa met MEL/J cellen die mazelen-H expresseren) 2. Correlatie IgG (MIA) tegen virus neutralisatie (VN): klassieke PRN test, tegen edmonston (conform 24 culture WHO protocol, 3rd WHO serum standard) F H PRN RIVM/EMC data 2016

MIA(IgG) > correlation with commercial EIAs ? Immune status HCWs (hospital survey) Vaccinated birth cohorts 10..20% seronegative/indeterminate different EIA tests, similar outcome JMV paper 2015; selectie van 154 HCWs, geb. jaren 1960-1995 EIA testen; Siemens/Enzygnost (wereldwijd meest gebruikt), en nog 2 klinische testen (Liaisson, Vidas, hier niet weergegeven) PRN: klassieke PRN test, tegen edmonston (conform 24 culture WHO protocol, 3rd WHO serum standard) < 5% seronegative with MIA Dorigo et al. 2015

Virus neutralizing antibodies Immune status HCWs (hospital survey) < 1% seronegative with PRN JMV paper 2015; selectie van 154 HCWs, geb. jaren 1960-1995 EIA testen; Siemens/Enzygnost (wereldwijd meest gebruikt), en nog 2 klinische testen (Liaisson, Vidas, hier niet weergegeven) PRN: klassieke PRN test, tegen edmonston (conform 24 culture WHO protocol, 3rd WHO serum standard) Dorigo et al. 2015

measles serostatus: notes and challenges Population-wise, majority of vaccinated persons have neutralizing antibodies Commercial EIA tests lack sensitivity, to detect protective antibodies in vaccinated persons (>10%) Better correlation between PRN and MIA (Luminex), not absolute Challenges: Alternative serological tests/indicators for serological comparisons Investigate different vaccine lots/regimens/programmes Testing of low-titre sera and seronegatives (LLOQ) Improve PRN testing >development high-throughput (96wells/FRN) format

Reducing the age of 1st MMR vaccination (6-12 months) new studies & plans (1) Reducing the age of 1st MMR vaccination (6-12 months) MIA children ‘at risk’ during latest measles epidemic in Biblebelt/The Netherlands immunization offered between 6 and 14 months of age clinical & immunological efficacy (2013-2017 trial/study) 6-8 months 9-12 months 14m/ control n= 39 31 37 Geometric mean (IU/ml) 0,7461 2,094 2,147 <0,12 IU/ml 3 Primary immunization at 6-8 months results in decreased GMTs compared to primary immuization at 9-12 or 14 months Preliminary data Brinkman et al. 2016

new studies & plans (2) New large population-based survey in The Netherlands 2016 (Pienter-3) Systematic analysis of IgG (MIA) and PRN in immunized persons from different vaccinate regimes/programs/lots Extend analysis to heterologous (wildtype) measles strains (EMC/RIVM) Serving LabNet with high-throughput Luminex-based MMR serology Capacity building for multiplex (MIA, FRNT) based serology for measles and mumps (EMC/RIVM)

Acknowledgements Fiona van der Klis Gaby Smits Iris Brinkman Nynke Rots Jeroen Kerkhof Hinke ten Hulscher RIVM/Virology & Immunology Lab Bilthoven, The Netherlands Rik de Swart Virosciences/EMC Rotterdam, The Netherlands

Seroprevalence HCW study EIA brand (manufacturer) # tested result PRN <120 PRN≥120 % seropositive*   all 1960-1974 1975-1985 1986-1995 Enzygnost (Siemens) 154 positive 139 90.8 31/31 (100%) 56/67 (84%) 52/56 (93%) negative 1 14 Vidas (bioMerieux) 137 89.5 55/67 (82%) 51/56 (91%) 16 Liaison (Diasorin) 136 88.9 54/67 (81%) 17 MIA (Luminex, inhouse) 149 97.4 63/67 (94%) 55/56 (98%) 4 Dorigo et al. 2015