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Case Study // JUN 8 2015

Setting the Foundation for an Ebola Vaccine

Generation of a cell line that is biologically contained for Ebola virus research

"The VeroVP30 cell line was established by cotransfecting Vero cells with pCAG-VP30 (for the expression of VP30) and pPur, a protein expression plasmid for the puromycin resistance gene (Clontech), using the transfection reagent TransIT®-LT1 (Mirus)..... To artificially generate EBOV, we transfected 5 x 105 293T cells with 1.0 µg of pTM-EbolaΔVP30, 2.0 µg of pCAG-L, 1.0 µg of pCAG-NP, 0.5 µg of pCAG-VP35, 0.5 µg of pCAG-VP30 and 1.0 µg of pCAG-T7 pol using TransIT®-LT1 (Mirus) in BSL-4 containment"

- Halfmann, et al.1

Ebola Virus Disease, or Ebola Haemorrhagic Fever, is caused by Ebola virus infection and is characterized by weakness, muscle pain, heavy internal and external bleeding and high fever. Symptoms occur on average about 8 days after infection and can lead to lethality in an average of 50% of infected individuals. The 2014 Ebola virus outbreak in West Africa was the largest in history. It was concentrated in Guinea and Sierra Leone but eventually spread to Liberia, Nigeria, Senegal, Spain, the United States, Mali and the United Kingdom. Deaths from the virus were estimated to have surpassed 10,000 individuals and at the time of the outbreak, there were no vaccines or specific treatments available. Largely as a result of a cooperative policy of containment, the World Health Organization finally called an end to the outbreak on May 9, 2015.

In this case study, Marzi, et al.2 built upon the work of Halfmann et al.1 and demonstrated that a replication defective form of the Ebola virus produced in Vero cells can effectively protect nonhuman primates from a lethal infection dose of Ebola virus.

1 Generation of biologically contained Ebola viruses
Halfmann P, Kim JH, Ebihara H, Noda T, Neumann G, Feldmann H and Kawaoka Y. PNAS, 2008 January 29, 105 (4): 1129-1133

2 An Ebola whole-virus vaccine is protective in nonhuman primates
Marzi A, Halfmann P, Hill-Batorski L, Feldmann F, Shupert WL, Neumann G, Feldmann H and Kawaoka Y. Science, 2015 April 24, 348 (6233): 439-442

Background:

In 2008 researchers in Dr. Yoshihiro Kawaoka's lab set about creating a system whereby the Ebola virus could be studied in a safer environment. To do so they created a Vero E6 cell line that stably expressed the VP30 gene under puromycin selection (VeroVP30). The VP30 gene is a component of the nuclear capsid and is absolutely necessary for viral replication and transcription. This stable cell line then was able to produce protein in trans to provide complementation to the other components of the Ebola virus genome, including a neomycin replaced VP30 (ΔVP30), to create viruses that resemble wild-type in their life cycle, morphology and growth properties but that are non-infectious and can be handled outside of a BSL-4 laboratory. As such, they created a biologically enclosed system for the study of the virus1.

The Mirus Bio TransIT®-LT1 transfection reagent was introduced in 1996 and still serves as a standard by which other reagents are measured. TransIT®-LT1 was the very first transfection reagent developed to be compatible with serum and is known for maintaining a low toxicity profile while providing robust DNA delivery in various cell types. TransIT®-LT1 has proven especially useful for virus production and was the reagent of choice by Dr. Kawaoka's lab for making the Ebola virus contained system.

Can a more effective vaccine be developed against the Ebola virus?

In the present case study, Marzi et al.2 described three different vaccine platforms that have been taken to clinical trials by other groups. The first, relies on DNA that expresses different Ebola virus glycoproteins. This has shown to be effective in nonhuman primates but requires multiple dosages of the vaccine in combination with recombinant adenovirus and has not been used as a standalone vaccine. The second is based on a chimpanzee adenovirus that expresses Ebola virus glycoproteins while remaining incapable of replication. This approach requires high doses and boosting to achieve extended protection for nonhuman primates. The third approach to reach the clinic is based on live-attenuated vesicular stomatitis virus expressing the Ebola virus glycoproteins. This approach affords complete protection in non-human primates but has increased potential risk of recombination due to the use of a replicating recombinant virus.

Marzi et al.2 believed they could provide a whole-virus vaccine, based on their earlier work producing a replication-defective Ebola virus1, that would provide distinct advantages over the three previous approaches to reach the clinic. Whole-virus vaccines have the advantage of providing multiple avenues by which the host immune system may recognize and mount a response against the virus by targeting multiple viral proteins or by degrading genetic material itself. The vaccine they developed is named EBOVΔVP30 and correlates with the Ebola virus lacking the VP30 replication capability.

To demonstrate the efficacy of EBOVΔVP30, Marzi et al.2 injected nonhuman primates, cynomolgus macaques, with 107 focus-forming units of vaccine in either one dose or two doses four weeks apart with appropriate controls. Four weeks after the last immunization, all animals were given a lethal dose of EBOV. Each of the four animals in the test groups were considered 100% effectively protected. It appears that immunization with EBOVΔVP30 causes an immune response primarily against the glycoprotein content of the virus affording protection to the animal. There was no appreciable immune response generated against the virus in control animals. In addition to the glycoproteins, they were able to find elevated antibody levels generated against the viral matrix protein (VP40) and nucleoprotein (NP) suggesting that the whole-virus vaccine was effective in providing a robust immune response. In addition to elevated antibody levels, test animals also had elevated numbers of interferon-gamma producing mononuclear cells relative to the control animals suggesting a better immune response.

The group wanted to increase the biosafety characteristics of the vaccine by treating with hydrogen peroxide (H202). They note that hydrogen peroxide acts to create breaks in single- and double-stranded DNA or RNA and thus could further inactivate a virus by preventing possible recombination events while preserving antigenicity. With n=2 animals, hydrogen peroxide treatment of the virus prior to two immunizations with virus also provided protection against a lethal dose of the virus suggesting that this is a further way to increase the biosafety of the vaccine.

In summary, generation of a cellular system in which Ebola virus could be more safely studied has led to a candidate Ebola virus vaccination treatment that appears to have enhanced efficacy and safety over other proposed approaches. A Mirus Bio transfection reagent, TransIT®-LT1, was chosen to create the biologically contained system and to produce high titer whole-virus in a replication incompetent form. Hopefully these advances will provide an avenue of protection for those that are stricken with this deadly disease.

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