A recombinant vaccine against the H5N1 influenza virus

1. A recombinant vaccine against the H5N1 influenza virus Presented by: Steven Mitchell

2. Background • Influenza is an infectious disease that arises within mammals and birds. • The influenza virus is categorized into three groups: • Influenza A: infects birds, swine, and humans • Influenza B: infects seals and humans • Influenza C: infects swine and humans • Influenza is named using the system H_N_ H for hemagglutinin; N for neuraminidase • Traditional vaccinations cover only H3N2, Influenza B, and H1N1 strain variants

4. Problems with traditional vaccines • Not all infectious agents can be grown in culture • Production of animal/human viruses require animal cell culture, which is expensive • Yield and production rate of viruses is relatively low • Batches of vaccine may not be killed, leading to inadvertent disease transmission. Attenuated strains may revert, and so constant testing is needed • Most vaccines have a limited shelf life and require refrigeration

5. Recombinant vaccine to prevent H5N1 • HPAI-H5N1 (bird flu) is an influenza A subtype virus that is highly pathogenic and has a high mortality rate • Current efforts toward a vaccine have been unsuccessful due to traditional methods unable to handle the rapid mutation of hemagglutinin (HA), a diverse surface protein present on all influenza viruses • Recombinant DNA technology may provide a preventative treatment for this pathogen

6. Diagram of an influenza virus

7. Principles of H5N1 recombinant vaccine • The Fc portion of IgG is an important fusion tag for expressing several viral proteins, such as SARS and HIV • The Fc portion helps folding of the viral protein to enhance binding of antigen-presenting cells. The Fd (foldon sequence of T4 phage fibritin) sequence was also expressed to further promote proper folding • Two vaccines were created by hybridizing the HA protein to either the Fc sequence of IgG, or Fc plus Fd

8. Structure of HA protein and recombinant Fc and Fdc protein The HA protein of H5N1 consists of a signal peptide, HA1, HA2, and a protease cleavage sites The SP sequence was replaced by IL2ss sequence in the Fc vector to create HA1-Fc protein HA1-Fdc was created by attaching HA1 to the Fd C-terminus, followed by the Fc vector

9. Results • Transfected HEK 293T cells were used as expression vectors for HA1-Fc and HA1-Fdc proteins • 293T cells were used because of their ability to amplify transfected plasmids. Other cell lines could be used for substitution (such as CHO cells) • Proteins were extracted from the supernatant medium, purified, then analyzed by SDS-PAGE and a western blot

10. Schematic of a subunit vaccine

11. Western blot for HA1 fusion protein The purified HA1-FC and HA1-Fdc proteins were analyzed using a western blot with an anti-HA mAb. Purified proteins were separated by 10-20% Tricine gels and transferred to nitrocellulose. Proteins were blocked overnight, then incubated with anti-HA mAb for 1 hour. Blots were incubated with HRP-conjugated goat anti-mouse IgG for 1 hour.

12. Results • Mice injected with the HA1-Fc and HA1-Fdc recombinant proteins were analyzed via ELISA for antibody responses • Both proteins induced IgG antibody responses specific to the proteins • Subsequent injections with different clade’s HA proteins showed an antibody response (pseudoviruses)

13. IgG antibody response to HA1-Fc and HA1-Fdc vaccine treatments Left: Reactivity of IgG antibody with HA1-Fc and HA1-Fdc proteins. Time scale was 10 days between first vaccination and boosters Right: The ability of IgG to bind to the proteins and the control

14. Results • The antibodies produced were found to reduce the viruses pathogenic qualities such as reproductive capabilities • The antibodies were effective for a wide array of live H5N1 strains, as well as H5N1 pseudoviruses, including: • HK/156 • VN/1194 • SZ/406H • HK-HA • 1194-HA • QH-HA • XJ-HA • AH-HA • These three avian flu strands are highly pathogenic in humans

15. Antibodies present from heterologous strains of H5N1 Graphs show various strains of live influenza being introduced into mice after being vaccinated. The neutralizing antibodies, NAb, block biological effects the virus has on it’s host cells. Hemagglutination antibodies, HI Ab, disable the binding ability of the influenza virus.

16. Viral challenge in vaccinated mice Left: Lethal H5N1 virus challenge in vaccinated mice. Three different clades were tested for 21 days. Note the difference in survival rate between proteins HA1-Fc and HA1-Fdc Right: Detection of viral RNA copies by quantitative reverse-transcription PCR (Q-RT-PCR) in lung tissues of H5N1 challenged mice

17. Histopathological changes in lung tissue Lung tissue samples were collected five days after all mice were sacrificed. All sections were stained with hematoxylin and eosin and observed under light microscope. Tissues are fixed in paraffin wax.

18. Overview • Targeting HA, the main surface protein of the virus, would provide a feasible means of producing an effective virus • The HA gene was fused with the Fc of IgG antibodies, or with Fc and Fd (foldon) to promote trimeric folding • Both subunit vaccines were shown to be effective for a wide variety of avian influenza strains. However, the HA1-Fdc vaccine proved to be the most effective

19. Methods and Materials • Construction, expression, and purification of recombinant HA1-Fc and HA1-Fdc proteins • Genes encoding HA1 of H5N1 were amplified by PCR using full-length HA as the template and inserted into the Fc expression vector. The Fd sequence derived from bacteriophage T4 fibritin was fused to the C-terminus of HA1 sequences by PCR with overlapping primers. The recombinant proteins were expressed in 293T cells using calcium phosphate method. The recombinant proteins were purified by protein A affinity chromatography. • Western Blot • The purified proteins were analyzed by SDS-PAGE and western blot using an anti-HA mAb. The proteins were transferred to nitrocellulose membranes. After blocking, blots were incubated with HA specific mAb for 1 hour. Blots were incubated with HRP-conjugated goat anti-mouse IgG for 1 hour. Signals were visualized with ECL reagents. • ELISA • The antibody response was evaluated by ELISA in collected mouse sera. 96-well ELISA plates were coated with recombinant HA1-Fc and HA1-Fdc fusion proteins, HA1 protein without Fd and Fc, and inactivated H5N1 virus and blocked overnight with non-fat milk. Bound antibodies were incubated with HRP-conjugated goat anti-mouse IgGfor 1 hour. The reaction was visualized by TMP and stopped by 1N H2SO4.

20. Further information • http://www.ted.com/talks/seth_berkley_hiv_and_flu_the_vaccine_strategy.html • http://www.who.int/csr/disease/avian_influenza/country/cases_table_2010_12_09/en/index.html • Barry, John. The Great Influenza. New York: Penguin Publishing, 2005. Print. • Shanks, Dennis, and John Brundage. "Pathogenic Responses among young adults during the 1918 Influenza Pandemic." Emerging Infectious Diseases. 18.2 (2012): 201-207. Print. <www.cdc.gov/eid>.

21. References • Davidson, Michael. "The Influenza (Flu) Virus." Molecular Expressions. Florida State University, 28 2005. Web. 3 Dec 2012. <http://micro.magnet.fsu.edu/cells/viruses/influenzavirus.html>. • Du, Lanying, Lanying Du, et al. "A Recombinant Vaccine of H5N1 HA1 Fused with Foldonand Human IgG Fc Induced Complete Cross-Clade Protection against Divergent H5N1 Viruses ." PLoS ONE. 6.1 (2011): 1-10. Print. • Glick, Bernard. Molecular biotechnology: principles and applications of recombinant DNA. 4th ed. Washington, DC: ASM Press, 2010. 459-497. Print. • Neumann, Gabriele, Hualan Chen, et al. "H5N1 influenza viruses: outbreaks and biological properties." Cell Research. 20.1 (2010): 51-61. Print. • Racaniello, Vincent. "Influenza Hemagglutination Assay." Virology Blog. N.p., 27 2009. Web. 3 Dec 2012. <http://www.virology.ws/2009/05/27/influenza-hemagglutination-inhibition-assay/>.