1. “Neutralizing Antibodies Derived from the B Cells of 1918 Influenza Pandemic Survivors” (Yu et. al) Daniel Greenberg

2. The Influenza Virus Negative sense single-strand segmented RNA virus Human influenza A and B cause seasonal epidemics (influenza C causes minor respiratory epidemics) Two main surface receptors: Hemagglutinin (HA) and neuraminidase http://micro.magnet.fsu.edu/cells/viruses/images/influenzafigure1.jpg

3. 1918 Spanish Flu Influenza A H1N1 virus of avian origin Most devastating epidemic in recorded world history Killed 20-40 million people Average U.S. life span lowered by 10 years http://www.whale.to/a/spanishflu.gif

4. 1918 Spanish Flu (cont.) Genome recently sequenced (8 genes of interest) Allows for molecular-level research (e.g. antibody-surface antigen interaction), not just serology http://www.proteinsciences.com/Catalog/files/assets/basic- html/page11.html

5. Question Do survivors of the 1918 H1N1 influenza virus show adaptive (acquired) immunity to the virus? 1918 H1N1 Survivor B Cells Antibodies 1918 Influenza Virus Reaction? http://blog.sciseek.com/wp- content/uploads/2009/12/swine -flu-h1n1-virus.jpg

6. Methods 1.Create monoclonal antibodies (mAbs) 2.Genetically sequence antibodies of interest 3.ELISA test 4.Hemagglutination inhibition assay (HAI) 5.Virus neutralization assay 6.Classify escape mutants and determine the mAbs to which they have resistance 7.Test for therapeutic efficacy using in vivo methods

7. Creating mAbs http://www.epitomics.com/images/RabMab-Flow-Chart.jpg 1918 H1N1 Survivor Myeloma cells

8. ELISA Test http://www.virology.ws/wp-content/uploads/2010/07/elisa.jpg Test used to determine whether an antibody is specific to a certain antigen (HA) Hybridoma Lines: 1I20 1F1 2B12 4D20 2D1

9. Antibody Gene Sequence V = “variable” region D = “diversity” region J = “joining” region H = heavy chain L = light chain Light chain CDRs important for binding to HA

10. ELISA Test Reaction with 1918 strain Cross-reaction with 1930 strain Minimal reaction with 1943/1977 strains (1F1)

11. Hemagglutinin Inhibition Assay (HAI) Test for inhibition of viral binding by antibodies

12. Neutralizing Assay Test for loss of infectivity of virus through reaction with an antibody > = No loss of infectivity at any concentration below 2.5 µg/mL

13. Classifying Escape Mutants (using HAI)

14. Tests for Therapeutic Efficacy

15. Implications B cells survive for life of host Memory B cells can also be long- lived Higher prevalence of viruses related to 1918 H1N1 may have led to long- term immunity High somatic mutation rate, as seen in mAbs, is associated with strong binding constants and high potency

16. Limitations Difficult to tell if isolated mAbs were first stimulated by the 1918 virus Limited sample size (only 32 subjects), so may not be representative of entire population http://www.chromatographytoday.com/assets/file_store/pr _files/22000/images/thumbnails/360w-new-antibody--can- neutralise-influenza-viruses-in-a-unique-way- _3194_801450250_0_0_14027843_300.jpg

17. Future Studies How does the 1F1 mAb bind to the 1977 strain if that strain’s Sb antigenic site is so genetically different from that of the 1918 strain? Further investigation into these mAbs could lead to development of therapies for a 1918-like virus Antibodies isolated from a person may give insight into the diseases they have been exposed to in their lifetime

18. References http://www.cdc.gov/flu/about/viruses/ty pes.htm http://virus.stanford.edu/uda/ http://www.sciencemag.org/content/31 0/5745/28.full http://pathmicro.med.sc.edu/mayer/igg enetics2000.htm