Dr. Lee C. Smith BEBPA, Basel 26 Sept 2013 Topics Covered What is - PowerPoint PPT Presentation

The Experimental Approaches Applied to Optimise and Control a Cell-based Potency Assay used to Test a Live Attenuated Dengue Vaccine Dr. Lee C. Smith BEBPA, Basel 26 Sept 2013

Topics Covered • What is Dengue and the need for a vaccine • The bioassay potency readout for DENVax • The approach taken in assay development • Selection & screening of assay parameters • Optimisation of assay set points • Assay precision analysis & assay transfer • Assay preparation for phase III validation

Dengue Fever Tropical/sub-tropical disease 3

Dengue Fever Dengue is “the most important mosquito - borne viral disease in the world” affecting populations across Asia, Latin America and Africa 2 Estimated annual global burden of Dengue • 400 million people infected • 100 million develop clinical illness Annual infections In 2010 • 500 thousand hospitalized • 20 thousand deaths, mostly in children

Dengue Fever  Over one half of world population threatened  70 – 500 million dengue infections per year  36 million cases of dengue fever per year  2.1 million cases of dengue hemorrhagic fever  > 20,000 deaths per year  Mosquito-borne infection  Four serotypes: DEN-1 through DEN-4  No therapeutic options  No vaccine currently available  Any vaccine candidate must protect against all four dengue serotypes 5

Zoonotic virus 1. Dengue fever 2. Dengue haemorrhagic fever * *Ab dependent enhancement (ADE)

4 Serotypes; all cause disease Any vaccine candidate must protect with neutralizing Abs against all four dengue serotypes 7

Measuring Vaccine Potency 8

Plaque Based Assays • Plaque assays - standard for virus concentration • A confluent cell monolayer is infected with the virus & covered with a semi-solid medium to prevent the virus infection from spreading indiscriminately • A viral plaque is formed when a virus infects a cell within the fixed cell monolayer which will lyse and spread the infection to adjacent cells where the infection-to-lysis cycle is repeated • The infected cell area will create a plaque (an area of infection surrounded by uninfected cells) which can be seen visually or with an optical microscope after 1 – 2 weeks 9

Immuno Focus Assay (IFA) • Rapid variation of plaque assay • Uses immunostaining with antibodies specific for the viral Ag • Measure host cell infection before an actual plaque is formed. • Shorter incubation period (e.g., 1-5 days) • Stain with 2 ° Ab enzyme conjugates • Substrate leads to coloured foci • Results are expressed as F ocus F orming U nits or FFU/mL 10

Spearman-Karber => & Reed-Muench 11

Dengue Immuno Focus Assay (IFA) • Relatively slow progress to distinct CPE • Stain virus plaques with anti-Dengue Abs after few days • Count Focus Forming Units (FFU/mL)

A Perfect Spot? 13

• We’re counting spots so we wish them to be: – Clean round spots – Separated from others – Unambiguous – The Right Size 14

Imperfections Comets Too small  15 Satellites Too big

Inaccuracy & An inability to count = the foci imprecision “The method needs to produce foci that are the right size, not merged, have no comets or satellites regardless of who performs it, where it is performed and when it is performed” 16

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DoE brings understanding – which parameters have an effect upon the desired responses – which parameters interact – which parameters need to be focused upon and optimised and those that can be fixed – The design space & how close to failure we operate the assay 18

System Variables Controllable (X) Factors System Response Measures Uncontrollable (N) Factors 19

Scoping - Assay Deconstruction PBS Wash Overlay Vol Pressure type Repeats Overlay conc Dilution series Temp Incubation Volume Time added Temp Blocking CO2 Vol conc conc Time Temp Mixing 2 o Ab Titer Comets Optional storage point Satellites Agitation Incubation Methanol fix Time Vol temp Stop trigger with wash Temp vol CO2 Time conc Inubation time Time uncovered Conc Vol AP Conc Agitation Repeats 20

Discuss & Agree what’s important 21

Risk Assessment Control Strategy Heat Map Unit Operation Method Parameters Ranges or Set Point CNX Accuracy Precision Foci size, comets & satellites Priority or Risk Overlay Incubation (Time) 4 – 7 days X 9 9 9 9.00 Overlay Type CMC vs Avicel C 9 9 5 7.40 Plate & Sample Prep Confluency 80-100% C 7 9 5 6.80 Plate & Sample Prep Passage number Currently at 149 C 7 7 5 6.26 Plate & Sample Prep Mixing/pipetting Mixing time 5 - 30 secs X 9 9 3 6.24 Virus adsorption Time dry (from aspiration of 60 - 300 secs N 9 9 3 6.24 Virus adsorption Sample Addition (Vol) 100 – 500 μl X 9 9 3 6.24 Overlay Concentration 0.4 – 1.0% X 7 7 3 5.28 Signal Development Incubation Time 20-90 mins C 7 7 3 5.28 Virus adsorption Incubation (Time) 1.5 – 24 hours X 9 5 3 5.13 Plate & Sample Prep Plate manufacturer Currently Greiner, cat. No. 657160 C 5 5 5 5.00 Virus adsorption Incubation (Temp) 35-38oC C 5 5 3 4.22 Virus adsorption Agitation Rocking vs Manual X 5 5 3 4.22 Plate & Sample Prep Plate Age 1.5 - 2.5 days C 7 9 1 3.98 Signal Development Incubation Temp 22-27oC N 7 7 1 3.66 Plate Fixing Wash repeats 2 - 4 C 3 5 1 2.47 Plate Fixing Wash agitation 6-10 swirls N 3 5 1 2.47 Plate & Sample Prep Dilution Series Factor 2 to 5 C 9 1 1 2.08 Virus adsorption Incubation (CO2 Conc) 3.5-6.5% C 3 3 1 2.08 Overlay Incubation (Temp) 36-38oC C 3 3 1 2.08 Immuno-staining Wash repeats 1 to 3 C 3 3 1 2.08 Immuno-staining Wash volume 1-3 mL C 3 3 1 2.08 Immuno-staining Blocking temp 25-40oC C 3 3 1 2.08 Immuno-staining Blocking Time 15-60 mins C 3 3 1 2.08 Immuno-staining Blocking volume 0.5-2mL C 3 3 1 2.08 Immuno-staining Primary Ab Vol 0.8 - 1.2 mL C 3 3 1 2.08 Immuno-staining Primary Ab Conc 1:1000-1:3000 X 3 3 1 2.08 Immuno-staining Primary Ab incbtn time 1-6 hr X 3 3 1 2.08 Immuno-staining Secndry Ab Vol 0.4 - 0.6 mL C 3 3 1 2.08 Immuno-staining Secndry Ab Conc 1:500-1:2000 C 3 3 1 2.08 Immuno-staining Secndry Ab incbtn temp 35 - 39 ºC C 3 3 1 2.08 Immuno-staining Secndry Ab incbtn time 30-120 mins C 3 3 1 2.08 Signal Development AP Volume 0.3ml-0.5mL C 3 3 1 2.08 Plate Fixing Methanol fix time 20-60 mins C 1 1 3 1.44 Plate & Sample Prep pH 6.8 - 7.6 C 1 1 1 1.00 Overlay Incubation (CO2 Conc) 4-6% C 1 1 1 1.00 Overlay Volume 2-4mL C 1 1 1 1.00 22

Overlay Adsorption Medium time 7 Overlay Inoculum Concentration volume Incubation Agitation time Mixing method 23

Screening Designs Selected 2 level fractional factorial with CP for curvature Parameters were a mix of Categorical (binary) or Numerical 24

Serotype 1 Screening data Half-Normal Plot H a lf-N o rm a l % P ro b a b ility H a lf-N o rm a l % P ro b a b ility 99 99 E-Mixing method BC 95 95 DE 90 90 B-Adsorption time BD 80 C-Overlay concentration 80 D-Incubation time D-Incubation time B-Adsorption time DE 70 70 E-Mixing method 50 50 30 30 20 20 10 10 0 0 0.00 384791182.00 769582364.00 1154373546.00 1539164728.00 0.00 4.10 8.20 12.31 16.41 20.51 24.61 |Standardized Effect| |Standardized Effect| Titer Comets Half-Normal Plot Half-Normal Plot H a lf-N o rm a l % P ro b a b ility H a lf-N o rm a l % P ro b a b ility 99 99 F-Overlay medium C-Overlay concentration 95 95 D-Incubation time 90 90 A-Infection volume B-Adsorption time 80 80 BD 70 70 50 50 30 30 20 20 10 10 0 0 0.00 0.34 0.67 1.01 1.35 0.00 4.20 8.39 12.59 16.78 |Standardized Effect| |Standardized Effect| Satellites Foci Size 25