Evaluation of Gene Copy Number in Vaccines Ali Azizi, Ph.D. Department of Analytical Research & Development Sanofi Pasteur

Disclaimer Views expressed in this presentation are those of the author and do not necessarily represent the opinions of Sanofi Pasteur (SP) or its affiliates. Any mention of commercial products within this presentation is for information only; it does not imply recommendation or endorsement by SP, nor does it imply that the products are the best available.

Outline Introduction -Genetic Stability -HSV candidate vaccine (HSV529) and AV529-19 cell line -Molecular methods Results -Evaluation of gene copy numbers -Intermediate Precision -Accuracy -Genome equivalence Take-home messages

Why Genetic stability is important? Genetic instability of the genes or transgenes within a vaccine may lead to inconsistent expression of complementing gene products and altered cellular properties. Therefore, it is important to demonstrate consistent and stable gene copy numbers over the generation length of a production run.

HSV-2 is widely recognized as a serious problem Worldwide, among 15 to 49 year-olds: 16%, or over 530 million people, are infected with HSV-2 and there are 24 million new infections each year In 2016, direct and indirect costs of HSV-2 infection are projected to reach over $2.5 billion/year in the USA alone Neonatal herpes  ~33% of cases cognitive impairment, severe neurological disease, organ dysfunction, or death Antivirals (e.g., Valtrex®) are not 100% effective and do not protect against infection HSV infection increases risk of HIV acquisition ~ 3x WHO urges rapid progress of an HSV vaccine 5

Description of our HSV-2 candidate vaccine (HSV529) Two essential genes(UL5 AND UL29) were deleted from HSV-2 vaccine candidate. Incapable of replication while preserving its ability to elicit a protective immune response in animal models. Two doses protect mice and guinea pigs against latent infection. HSV529 vaccine

X AV529-19 cell line No Replication Infect Infect AV529-19 cell line No UL5 or UL29 Infect AV529-19 cell line Infect HSV529 Vaccine UL5 AV529-19 is a Vero cell line specifically engineered to express the HSV-1 UL5 and UL29 transgenes UL29 HSV529 Vaccine

HSV529 confers better protection versus gD subunit vaccine in guinea pigs (HSV-1 pos)

Rationale of developing HSV529 Feature Live-Attenuated gD2 Vaccine HSV529 Replication Defective Antibody titers ?   MHC I and II presentation T-cell response No need for adjuvant Protection in guinea pigs

Key features before human trials TEST Cell Seed MCB WCB ECB Morphology + Karyotype Life Span Sterility - Mycoplasma Spiroplasmas Stability Extraneous Agents Co-Cultivation Test in animal and Eggs Specific Tests on Contaminating Agents Retroviruses Tumorigenicity Ph. Eur. 5.2.3

Stability of AV529-19 cell line Genetic instability of the UL5 and UL29 genes may lead to inconsistent expression of complementing gene products and altered cellular properties of the AV529-19 cell line. Therefore, it is important to demonstrate consistent and stable UL5 and UL29 gene copy numbers in AV529 cells over the interval of cellular replication from MCB to end of production.

Is qPCR the best molecular method approach? AV529 cell line Extraction of DNA from AV529 cell lines. Design of customized PCR primers for in-house reference controls (GAPDH, B-actin) and test samples (UL5 and UL29). Generation of GAPDH and B-actin in-house reference standards Optimization of PCR amplification conditions for detection of either UL5 or UL29. Development and generation of DNA standard curves for relative quantification. Determination of the relative gene copy number by comparison between Cp values of test samples and in-house reference controls. DNA extraction

Challenges with qPCR -qPCR method is not able to provide an absolute quantitation of gene copies present per sample and requires a reference standard. Therefore, a qPCR-based method provides relative quantitation. -Difference in amplification efficiencies between the reference standard and test samples are another common confounding issue for qPCR-based methods. -The full length genome sequence of Vero is not available, therefore, it is hard to have a reliable standard control with a known copy numbers.

Digital PCR (dPCR) dPCR is a molecular approach (not very new, however, there have been some new developments in instrumentation and application of method) to nucleic acid detection and quantification. It changes the analogue signal of qPCR into a digital output. It directly counts the number of target molecules rather than relying on reference standards or endogenous controls. Analysis by Poission distribution “Bio-Rad website”

dPCR advantages No need to rely on references or standards. Therefore, it measures the Absolute quantification. Highly tolerant to inhibitors, capable of analyzing complex mixtures. Wide variety of applications including Copy Number Variation, Mutation Detection, and Gene Expression.

Objective -To determine the UL5 and UL29 gene copy numbers in the HSV529 lots using a digital PCR-based approach. Design and selection of primers Isolation and quantification of DNA from AV529 cell line Dilution of test samples prior to dPCR Testing preparation of dPCR master mixes Determination of fraction of positive amplification reactions Assessment of prior restriction endonuclease digestion of test sample Assessment of the accuracy of the dPCR method by comparing the theoretical copy number values versus actual copy number values. Optimization of assay

Overview DNA extraction Nanodrop PCR reaction 384 well plate Dilute Light Cycler480 DNA melting curve analysis Poisson distribution Average copy number of the target/reaction Fraction of positive wells Average amount of DNA (pg)/reaction Target copy number per cell Total DNA (pg) in each Vero cell (about 7.9 pg)

The QX200 has a simple workflow and 96-well throughput Make droplets Perform PCR with droplets Determine Target copies Read droplets

The effect of restriction endonuclease digestion on AV529-19

Evaluation of Intermediate Precision in AV529-19 ICH Topic Q 2 (R1) Validation of Analytical Procedures: “Intermediate precision expresses within-laboratories variations: different days, different analysts, different equipment, etc”. -To evaluate intra-laboratory variations, the intermediate precision was assessed by performing the assay 8 times by 2 analysts on different days.

Accuracy ICH Topic Q 2 (R1) Validation of Analytical Procedures: “The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found”. NAME OF PRESENTATION

ACCURACY & PRECISION Accurate and precise

Evaluation of Accuracy in AV529-19

Gene copy number in AV529-19 MCB and AV529-19 EOPC using 384 well plates

Genome equivalence of HSV529 by dPCR without DNA extraction Preparation of samples Assay Results Dilute HSV529 dPCR 0.95 x 10^8 Dilute/Triton X100 1.41 x 10^8  Proteinase K/SDS/HI/Dilution 3.6 x 10^8  Proteinase K/HI/Dilution 1.24 x 10^8 DNA extraction qPCR 4.6 x 10^8 NAME OF PRESENTATION

Take-home messages No references or standards are required with the developed assay while a reference is required with the traditional methods like qPCR. Unlike traditional methods, the developed digital PCR provides an absolute copy number. The dPCR assay is fast (1 day) with a high throughput (over 90 samples can be characterized in each run). The developed dPCR-based approach could be easily applied to other cell lines. Therefore, it opens a new window in the field of analytical sciences. Despite many advantages, dPCR has its own limitations (varies in number of partitions per run which affect absolute precision, accuracy,…) .

Acknowledgement Bryan McNeil Liam Sanio Eyal Yefet Lucy Gisonni-Lex Robert Ryall “And my colleagues in the Department of Analytical Research and Development at Sanofi Pasteur, Toronto”

THANK YOU. For further information, please email me at ali THANK YOU! For further information, please email me at ali.azizi@sanofipasteur.com

Poission Distribution (back up slide) The Poisson distribution is a well-known statistical discrete distribution. It expresses the probability of a number of events (or failures, arrivals, occurrences ...) occurring in a fixed period of time. In digital PCR the test sample is diluted such that some reactions have no target sequence present, while others have one or more targets present. After amplification, reactions with a target produce positive results and reactions without target yield negative results. Average copy number of the target/reaction Fraction of positive wells Target copy number per cell Average amount of DNA (pg)/reaction Total DNA (pg) in each Vero cell (about 7.9 pg) Life Technologies Website