Ebola Virus Vaccine Production

Ebola viruses are a group of highly pathogenic filoviruses that cause outbreaks of severe hemorrhagic fever in humans and non-human primates, with a rate of high mortality. This virus was first recognized in The Democratic Republic of Congo in 1976. Since its discovery, it continues to cause outbreaks in equatorial Africa. Ebola virus constitutes an important local public health threat in Africa, with a worldwide effect through imported infections and through the fear of misuse for biological terrorism.

A vaccine which is safe and effective is needed because of its continuous emergence. The second challenging issue for an Ebola virus vaccine is to have the ability to protect against infection from aerosolized viruses. This has been a long-time concern, as aerosols are the most likely form for these viruses as a biological threat. This study indicates the potential for developing an effective monovalent Ebola virus vaccine based on the cAdVax technology, which demonstrated effectiveness in protecting against direct infection and aerosolized infection.

These results show that it is feasible and of high importance to create an Ebola virus vaccine that would be effective in the event of a natural outbreak or the event of the virus being used as biological threat. Introduction Ebola virus is regarded as the prototype pathogen of viral haemorrhagic fever, causing severe disease and high case fatality rates. This high fatality, combined with the absence of treatment and vaccination options, makes Ebola virus an important public health pathogen and biothreat pathogen.

Ebola virus and Marburg virus constitute the family Filoviridae. Filoviruses are enveloped, non-segmented, negative-stranded RNA viruses of varying morphology. These viruses have characteristic filamentous particles that give the virus family its name [4]. The exact origin, locations, and natural reservoir of Ebola virus remain unknown. However, on the basis of available evidence and the nature of similar viruses, researchers believe that the virus is zoonotic with four of the five subtypes occurring in an animal host native to Africa.

A similar host, most likely in the Philippines, is probably associated with the Ebola-Reston subtype, which was isolated from infected cynomolgous monkeys that were imported to the United States and Italy from the Philippines. The virus is not known to be native to other continents, such as North America [4]. Ebola forms long filamentous virions inside infected cells. When a virion is made, the structural proteins associate with the RNA strand, packaging it in a capsid that then associates with viral proteins that insert into the cell membrane, which allows the whole package to bud off from the infected cell and form a new virion.

The genetic material is a single strand of antisense (-) RNA of about 20,000 nucleotides. When transcribed by its own polymerase enzyme, the viral RNA codes for a nucleoprotein, a few structural proteins, the polymerase, and a glycoprotein [3]. The glycoprotein forms spikes, approximately seven nanometers long, on the virion surface. These glycoproteins define the receptor specificity, mediate the cell fusion and cell entry, and may have certain domains that interfere with other cell functions. Like all viruses, Ebola has a certain cell specificity; it targets endothelial cells and macrophages.

Ebola may even use its spikes to spread from cell to cell, thus evading the immune system and increasing its virulence [3]. Which most likely has something to do with its extreme pathogenicity and the fact that the immune response to it is so slow. Once inside a cell, the virion uncoats and the polymerase transcribes the viral (-) RNA into a (+) sense strand inside a host cell’s cytoplasm. There, the sense strand, and at some point, the polymerase switch into replication mode and copy the (+) sense strand into an anti-(-) sense strand. These are packaged with other virus components and released, along with components of infected cells [3].

Infected cells release a storm of early cytokines, like TNF-alpha, interleukin-6, and the interferons alpha and beta. These cytokines are very toxic and cause shock and damage to the body. Death comes from a combination of dehydration, massive hemorrhaging, and shock, which results from this massive release of cytokines [3]. Our vaccine development strategy constitutes a benign infection caused by a complex adenovirus vaccine vector that is replication defective, with the antigenic potential conferred by highly induced expression of Ebola virus GP genes.

It is our hypothesis that de novo synthesis and expression of Ebola virus antigens will mimic the antigen presentation that would occur from a natural Ebola virus infection, but without causing pathogenicity and hemorrhagic fever associated with an actual Ebola virus infection. By mimicking Ebola virus infection, the presentation of Ebola virus antigen to the immune system should elicit an immune response against Ebola virus from both the humoral and cell-mediated parts of the immune system. In this study, we deveop a Complex Adenovirus Vector-based monovalent Ebola virus vaccine candidate.

This vaccine efficiently expresses the Zaire Ebola GP genes from the vaccine construct, which demonstrates effective induction both anti-Ebola virus GP serum antibody as well as Ebola virus-specific cell-mediated immune responses. Significantly, vaccination of non-human primates with the vaccine candidate led to 100% protection of the primates from any lethal challenge with Zaire Ebola virus. This induction of a protective immune response with 100% efficiency indicates the potential for developing an effective EBOV vaccine based on the cAdVax technology. Materials and Methods * Cell Lines

HEK293 (human embryonic kidney) cell lines were obtained from Coriell Institute for Medical Research Culture Collection. The cells were maintained in Dulbecco’s modified Eagle’s medium. HEK 293 cells were grown in a monolayer in flasks. Under optimum growth conditions (37°C, 5% CO2), 293 cells doubled about every 36 hr [1]. * Construction of the cAdVax EBOV vaccine The Ebola virus gene sequences included in the cAdVax vaccine were derived from the Zaire species. The Ebola virus GP genes were amplified by PCR, with each primer including specific restriction sites at the 5′ ends for subsequent cloning of the PCR fragments into shuttle vectors.

These genes were modified to delete the RNA editing signal responsible for initiating a secreted, nonstructural form of GP. Both genes were amplified by PCR and then subcloned into the plasmid shuttle vector. The CAdVax vector genome is devoid of E1, E3, and most of E4 (with the exception of ORF6) However, this vector is still capable of efficient replication in the standard human embryonic kidney 293 (HEK293) cell line, which provides E1 in trans[6]. Each CAdVax vector was propagated in HEK293 cells.

The vector was processed, followed by genome screening of vector clones for the correct transgene inserts using restriction mapping digestion, PCR, and DNA sequencing analyses. This ensured that no genetic deletions or rearrangements had occurred during the vaccine production steps. The lots of the final vaccine vectors were purified by ultracentrifugation and stored frozen in liquid nitrogen. The genomic DNA from the final vaccine vector was confirmed by restriction digest mapping, The modified virus containing the Ebola virus GP gene was then produced in the HEK293 cell line.

Protein expression from each vaccine component was confirmed by Western blotting, immunofluorescence assay, and immunogenicity was confirmed in primates. Fig. 1. Essential steps in generating an adenovirus vector to express a protein of interest. Cloning your gene of interest into a Shuttle Vector. Obtaining recombinant Adenoviral DNA with the gene of interest in the right orientation. Virus production in HEK293 or similar packaging cell line and preparation of a crude viral lysate. Amplification and Purification of the Adenovirus obtained. Titration of the virus [1].

ITR ITR hCMVie hCMVie FIG. 2. Strucure of adenovirus carrying Zaire Ebola Virus GP gene. ITR, inverted terminal repeat; hCMVie, human cytomegalovirus intermediate/early promoter; BGH polyA, bovine growth hormone polyadenylation site. * Animals Ten macaques were used in this study. They were targeted with a double dose of the vaccine over a period of ten days; and subsequently were infected with the Zaire Ebola virus. Five of the ten primates were infected with the Sudan Ebola virus strain. All animals were monitored closely over the period of 28 days.

Results Vaccination with the vaccine candidate provided protection against the Zaire strain of Ebola virus. All five of the primates vaccinated and then infected with the Zaire Ebola virus survived infection without any lethal challenge. However, primates infected with Sudan Ebola virus did not survive infection. The vaccine candidate proved to protect against one strain of the Ebola virus. Which suggests that each strain is distinct and protection against one strain of the Ebola virus does not offer protection against another.

Vaccination provided complete protection against aerosolized infection with Zaire Ebola virus. Reactive antibody titers were measured after each vaccine dose. Western blot analyses indicated that the vaccine induced expression of the GP. Discussion Currently, there is no preventative treatment against the deadly hemorrhagic fever caused by Ebola virus infection. This is because of the highly contagious and deadly nature of filoviruses, there is great concern that these lethal agents may be used as biological weapons or terrorism agents against humanity.

It is also feared that these viruses may spread into populated urban areas as a result of increased travel. Therefore, the development of effective Ebola virus vaccines to prevent the further evolution and spread of Ebola virus has become a great interest to many in the research community. In this study, we developed and evaluated a monvalent Ebola virus vaccine designed to prevent infection by the Zaire species. However, one major difficulty that remains in the development of an

effective EBOV vaccine is the requirement for a bivalent vaccine to induce protective immune responses against two Ebola virus species, Zaire and Sudan, which have been responsible for all human deaths due to Ebola virus infection thus far. In addition, our vaccine demonstrated 100% protection of the primates from any lethal challenge with Zaire Ebola virus. This induction of a protective immune response with 100% efficiency indicates the potential for developing an effective EBOV vaccine based on the cAdVax technology. The major advantage of the cAdVax system vector is the ability to express multiple antigens in a single construct.

Upon vaccination, all of the antigens carried by the vector will be produced at high levels within the cells transduced at the site of vaccination[6]. We hypothesize that vector-based vaccine gene transfer induces a de novo antigen synthesis, which results in a natural antigen expression and presentation on cell surfaces. This mimics a natural infection by the pathogenic viruses and induces potent immune responses without causing the disease. Vaccines based on antigen synthesis de novo create a major advantage over protein-based subunit vaccines that are only capable of presenting linear epitopes.

They also have an advantage over recombinant protein antigen synthesis in eukaryotic cells in which the correct conformation of the glycoproteins that contain the receptor-binding site may be destroyed in the extensive purification processes. In contrast, GP antigens synthesized de novo would theoretically retain the natural conformations and posttranslational modifications of the native GPs and therefore would include intact viral receptor-binding sites, where virus-neutralizing epitopes would be located[2,5]. In addition to antibody responses, the cell-mediated immune system is critically important in defense against virus infections.

Activated T lymphocytes play an essential role in destroying infected cells, preventing viral replication, reducing viral load, and eventually eliminating the infection. In the case of filovirus infections, mortalities often occur before sufficient time is allowed for the activation of cell-mediated immune responses. We hypothesize that activation of an EBOV-specific cell-mediated immune response prior to exposure to Ebola virus would give the cellular immune system a chance to establish itself and proliferate quickly in the event of an infection[2,5].

Among the many advantages of the cAdVax vaccine system is its ability to express multiple antigens in a single vaccine construct, thereby simplifying the production processes that would be necessary to bring a final Ebola virus vaccine to the public, which will be the direction of our future studies. In conclusion, our study suggests that a cAdVax-based vaccine, represents a promising candidate for the development of an effective monovalent vaccine against Ebola virus infections which we hypothesize, using the same technology will be effective in developing a bivalent vaccine against the two most lethal strains of the Ebola virus.

References [1] “Adenoviral Gene Expression Resource”. Clontech. 2010. Retrieved 2010-12-08. http://www. clontech. com/support/tools. asp? product_tool_id=152550&tool_id=154900 [2] Appaiahgari, M. B. , R. M. Pandey, and S. Vrati. 2007. Seroprevalence of neutralizing antibodies to adenovirus type 5 among children in India: implications for recombinant adenovirus-based vaccines. Clin. Vaccine Immunol. 14:1053–1055. [3] Bardi, Jason Socrates. “Death Called a River”. Scripps Research Institute . 2002. http://www. scripps. edu/newsandviews/e_20020114/ebola1.

html. Retrieved 2010-12-08. [4] “Ebola Hemorrhagic Fever Fact Sheet”. CDC. 2009. Retrieved 2010-12-08. http://www. cdc. gov/ncidod/dvrd/spb/mnpages/dispages/Fact_Sheets/Ebola_Fact_Booklet. pdf [5] Jones, S. M. , H. Feldmann, U. Stroher, J. B. Geisbert, L. Fernando, A. Grolla, H. D. Klenk, N. J. Sullivan, V. E. Volchkov, E. A. Fritz, K. M. Daddario, L. E. Hensley, P. B. Jahrling, and T. W. Geisbert. 2005. Live attenuated recombinant vaccine protects nonhuman primates against Ebola and Marburg viruses. Nat. Med. 11:786–790.

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