Viral Disarmament: A Trojan Protein Approach

O.M.I. & C.M.

July 28, 2015 – Genome Meeting

Project Overview• Project goal(s):

a) Is to study the reverse evolution of viruses. (i.e. loss in infectious ability)b) Is to decrease the virulence of viruses.c) We hope to determine whether therapeutics based on this approach may be potentially feasible.

• Project hypothesis:• Each virus will be passaged multiple times through a host cell line that is already expressing one viral

protein (called supplemented cells).• Our hypothesis is that the viral population will rapidly evolve to NOT produce the supplemented viral

protein. (viral adaptation / laziness)• If this occurs, the viral population will become replication deficient in wild type cells.

• Our project has 4 vital components:1. Learning how quickly a viral gene / genome can become non-functional2. Examining the effects of viral mutations on viral function (rates of viral evolution) 3. Determining the roles of viral genomic RNAs (vgRNAs) in viral replication and infection of host cells4. Examining viral mutations and whether the mutation preserves or causes a loss in function

RNA vs. DNA Viruses

This rapid evolution of viruses can be used against them….

Rapid mutation seen in RNA viruses

Inability of immune system to defend the host against them

Acute diseases: SARS, Influenza, Ebola, Yellow Fever…Chronic diseases: AIDS, Hepatitis C

Mutation rates: DNA viruses RNA viruses< 1:1000 to 1:100,000 per base/ per

generation

1:1,000,000 to 1:100,000,000 per base/ per generation

Due to lack of proofreading activity of RNA virus polymerases,

etc.

Difficulty producing efficient therapeutics / vaccines

Viral Protein - Supplemented MDCK Cells• Influenza A genome

comprises 8 different segments.

MDCK WT cells PB1C2 cells (PB1 IAV protein) M1C2 cells (M1 IAV protein) HAC9 cells (HA protein)

+MDCK WT cells

PFU

WT

/ P

FU V

P 1Infectivity

in WT cells?

Generation GNG0

ORIGINAL VIRAL SAMPLE Infection with

G0 (IAV – H1N1 Calif. 2009)

In-vitro Viral Evolution

Model

(Viral Protein -supplemented MDCK

cells)

Virus (Wt-generation x)

WT-Cells

Newly evolved viral population

(G1… GN)

RE-INFECT! Repeat for multiple

generations (G1, 2, 3…..)

Virus (Supp.Cell-generation x)

Newly evolved viral population

(G1… GN)

RE-INFECT! Repeat for multiple

generations (G1, 2, 3…..)

Measure % replication competent viruses.

If 0% end point is reached, SEQUENCE, SEQUENCE, SEQUENCE!

HARVEST HARVEST

INFECTINFECT

R & D Approach / Experiments Performed

• Selection process protocol / In-Vitro Evolution Model:a) Viral infection protocolb) Viral harvesting

• Cell culture:a) Cell growth and maintenance (passing, freezing, reanimating, etc.)

• Plaque forming unit (PFU) assay:a) Used in measuring the virus titer - concentration of viruses in a sample.

• Viral RNA extraction:a) Zymo Direct-zol™ RNA MiniPrep (works like a champion)

• qPCR (quantification):a) Flu-2 assayb) New TaqMan® NP6 qPCR assay (reduction in primer dimers / noise)

Sample In-vitro Evolution Infection Results (from 7/20/15 – 7/26/15)

Figure 1 - Pictures of cell morphology / cytopathic effect during the infection process, broken down in intervals of 24 hours.

Figure 1 Figure 2

Figure 2 – sample qPCR data from recent infection; initial and final samples taken from each flask. Δct calculated from different assays.

Recent Developments / Future Work

• What we have learned:• Optimizing supplemented cells growth & infection rates (vs. MDCK WT cells)

• Optimization of new TaqMan® assay for qPCR

• Optimization of Geneticin® (G418) concentrations for supplemented cells

• What we are currently working on:• M1C3 selection / in-vitro evolution process (currently on generation 3)

• M1C3 – G3 results are in the process of being sequenced, as well as WT – G3 & G1

• What is planned:• In-vitro evolution process for other clones (HA, PB1)

• Sequencing of results + Bioinformatic analysis / Mapping

QUESTIONS?Or if you want your immune system primed before flu season by hanging out with me in the lab (JK)

Thanks for your time!Special thanks to Momchilo Vuyisich,

Jennifer Harris, Murray Wolinisky, Jason Gans, Anais Weibel, Andy Hatch,

and Ryan Toma!