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

2. 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.

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

4. 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.

5. 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

6. 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

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

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

9. 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

10. 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

11. 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 AV 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.

12. 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

13. 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.

14. 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”

15. 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.

16. 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

17. 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)

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

19. The effect of restriction endonuclease digestion on AV529-19

20. 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.

21. 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

22. ACCURACY & PRECISION
Accurate and precise

23. Evaluation of Accuracy in AV529-19

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

25. 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

26. 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,…) .

27. 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”

28. THANK YOU. For further information, please email me at ali
THANK YOU! For further information, please me at

29. 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