Influenza Hemagglutinin

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Influenza, also commonly known as the flu, is a contagious respiratory illness caused by various influenza viruses that infect the nose, throat, and lungs []. Although influenza is typically a mild and common illness, it has caused 40,000 deaths and 100,000 hospitalizations annually in US. The seasonal influenza vaccine remains the most effective method to prevent the flu.

There are 3 types of viruses: A, B, and C. Type A and type B are the major pathogens in humans, which is why current influenza vaccines are trivalent, consisting of two influenza A subtypes (H1N1 and H3N2) and one or two variants of influenza B virus (Lee 2). Each year, the strains are chosen based on which strains of virus are predicted to most commonly circulate in humans each winter flu season.

While recent studies by the CDC show that the seasonal vaccine can reduce the risk of flu illness by approximately 50-60%, these “inactivated” vaccines only offer short-term and highly specific humoral immunity attributed to the frequent antigenic variations in the influenza virion (1). One solution to these issues is cold-adapted “live attenuated” vaccines that offer broad-spectrum immunity. One such conserved antigenic target is the hemagglutinin HA2 stalk domain. Proposed mechanisms of protection include non-neutralizing antibodies, inhibition of fusion of virus, maturation of the HA, and antibody dependent cell-mediated cytotoxicity (Lee 2).

Structure, function, and mechanism of Hemagglutinin

Hemagglutinin (HA) is a glycoprotein on the surface of influenza viruses that causes red blood cells to clump in the presence of an antibody (Figure 1 http://upload.wikimedia.org/wikipedia/commons/0/02/Flu_und_legende_color_c.jpg). It is approximately 13.5nm long and forms a cylindrical shape. Hemagglutinin contains three identical monomers from an alpha helix coil, which hold the sialic acid binding sites. Each of these HA monomers has two components: a long, helical chain by HA2 and a large HA1 globule (http://openi.nlm.nih.gov/imgs/512/37/3410141/3410141_pjab-88-226-g007.png).

HA1 and HA2 have different structures and mechanisms. HA1 holds the binding globular head domain and a conserved disulfide bond linking cysteine 52 and cysteine 277, which contribute to its stability (Steel). Current vaccines target the head domain surrounded by variable antigenic sites, but these vaccines have a low efficacy due to mutations of HA1. These mutations occur because of antigenic drift, small changes over time, or antigenic shift, abrupt and large changes. Antigenic drift is caused by frequent point mutations in HA and NA proteins in influenza virions due to lack of proof reading enzymes. The amino acid substitutions in these proteins makes the mutant virions go undetected by host antibodies. Antigenic shift occurs when the fragmented genomes of an influenza virus undergoes genetic variations when rare gene exchange occurs between human and non-human (e.g. avian, equine) viral strains, creating a new genotype and novel HA and/or NA proteins.

HA2 consists of the membrane-fusion inducing stalk domain that allows virus particles to enter. The N-terminus of HA2 holds the fusion peptide, which is hidden in the HA structure. This region cannot interact with the hydrophobic environment until the pH of the cell decreases, causing the structure to slightly unfold and expose the fusion peptide (Figure 2). The sequence conservation of HA2 (51-80%) is higher than that of HA1 subunit domain (34-59%) (Lee 2 84).

Influenza hemagglutinin has three main functions. One function is it binds the virus to its receptor on the target site. The HA1 globular head domain contains the sialic acid binding site, which binds to the sialic acid receptor on the target site. Another function is the ability of the virus to escape neutralization by the host due to antigenic variation. A third function is catalysis of membrane fusion. HA assists the entry of viral DNA into target cells by fusing the virus with the target cell (Perf. Stephen Harrison. Influenza Virus Particle. IBiology, 2011. Web. 19 Mar. 2015.).

File:File.http://upload.wikimedia.org/wikipedia/commons/0/02/Flu und legende color c.jpg

Structure of influenza virus. HA represents Hemagglutinin and NA represents neuraminidase.

Electron micrograph of the Ebola Zaire virus. This was the first photo ever taken of the virus, on 10/13/1976. By Dr. F.A. Murphy, now at U.C. Davis, then at the CDC.

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Legend/credit: Electron micrograph of the Ebola Zaire virus. This was the first photo ever taken of the virus, on 10/13/1976. By Dr. F.A. Murphy, now at U.C. Davis, then at the CDC.
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Section 2

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Examples of Universal HA Constructs

Vaccination of mice with headless HA constructs provides full protection from death and partial protection against disease following lethal viral challenge (Steel). Headless HA constructs also offer immune sera (make link to Wikipedia) with broader reactivity than full-length HA as well as cross-reactivity against several subtypes of HA.

Various vaccinations with variations of HA, including vaccinations that lack a globular head domain of HA1 but maintain the integrity of the stalk region, were created and injected into mice twice, on days 0 and 21. On day 77, all of the mice were challenged with a PR8 virus. All of the mice with the headless HA construct survived and showed a small percentage of average body weight loss. In comparison, 3 out of 4 of the mice with the full-length HA died because of the significant body weight loss (Figure: http://mbio.asm.org/content/1/1/e00018-10/F5.large.jpg).

These findings suggest that headless HA constructs are more effective because they attempt to avoid the highly immunogenic head domain. The survival of all the mice with headless HA constructs also shows promising results towards using headless HA as a universal vaccine.

File:Http://mbio.asm.org/content/1/1/e00018-10/F5.large.jpg

Vaccination of mice with headless HA constructs provides protection from death following infecting of mice with challenge with PR8 virus. Error bars indicate standard deviations and a star represents mouse death.

Further Reading

[Sample link] Ebola Hemorrhagic Fever—Centers for Disease Control and Prevention, Special Pathogens Branch

Lofgren, E., N. H. Fefferman, Y. N. Naumov, J. Gorski, and E. N. Naumova."Influenza Seasonality: Underlying Causes and Modeling Theories." Journal of Virology 81.11 (2007): 5429-436. Web of Science. Web. 24 Feb. 2015.

References

1 Bardiya, N., and J. H. Bae. "Influenza Vaccines: Recent Advances in Production Technologies." Applied Microbiology and Biotechnology 67.3 (2005): 299-305. Web of Science. Web. 24 Feb. 2015.

2 Lee, Young-Tae, Ki-Hye Kim, Eun-Ju Ko, Yu-Na Lee, Min-Chul Kim, Young-Man Kwon, Yinghua Tang, Min-Kyoung Cho, Youn-Jeong Lee, and Sang-Moo Kang. "New Vaccines against Influenza Virus." Clinical and Experimental Vaccine Research 3.1 (2014): 12. Web of Science. Web. 25 Feb. 2015.

3 "Selecting the Viruses in the Seasonal Influenza (Flu) Vaccine." Centers for Disease Control and Prevention. Centers for Disease Control and Prevention, 22 Sept. 2014. Web. 22 Feb. 2015.

4 Steel, J., A. C. Lowen, T. T. Wang, M. Yondola, Q. Gao, K. Haye, A. Garcia-Sastre, and P. Palese. "Influenza Virus Vaccine Based on the Conserved Hemagglutinin Stalk Domain." MBio 1.1 (2010): E00018-10. Web of Science. Web. 28 Feb. 2015.

5 Stephen Harrison. Influenza Virus Particle. IBiology, 2011. Web. 19 Mar. 2015

Edited by Gabriella Newman, a student of Nora Sullivan in BIOL168L (Microbiology) in The Keck Science Department of the Claremont Colleges Spring 2015.