1. Mechanisms of Severity of Pandemic H1N1 2009 Influenza A Virus Infection During Pregnancy, A Mouse Model Glendie Marcelin, PhD 3 rd World Congress on Virology November 21, 2013

2. Richard J. Webby, PhD Jerry R. Aldridge, PhD Adrianus C.M. Boon, PhD Jerold Rehg, PhD Henju Marjuki, PhD Susu Duan, PhD Jonathan A. McCullers, MD Hazem H. Ghoneim Flow Cytometry and Cell Sorting Ann-Marie Hamilton-Easton, PhD Perry Scott, PhD Veterinary Pathology Core Pamela Johnson Dorothy Bush Tetramer Core Facility Scott Brown, PhD Animal Resource Center David Carey Beth Little NIH NIAID WHO Global Surveillance Network Centers of Excellence for Influenza Research and Surveillance ALSAC Marcelin et al, J Virol PMID: 21865394 Trudeau Institute

3. Louie et al, N Eng J Med 2010 * * Compared to non-pregnant

4. Th1/Th2 T cell-Helper Responses During Pregnancy Pregnancy Basal Fetal tolerance Fetal rejection

5. Lung Injury and alterations in immune responses responsible for severity?

6. PNP Influenza A/California/04/2009 (H1N1): “pH1N1” Lung, Upper Respiratory, Liver, Kidney, Brain, Spleen CD8 +, CD4 + T cells T regulatory cells Antibody Cytokines Virus Replication Pathology Innate/Adaptive E13 Pathology Lung Repair Severity of Pandemic H1N1-09 in Pregnant Mice

7. *p= 0.01 10 6 EID 50 pH1N1 is More Severe in Pregnant Mice

8. Severity Not Due to Increase in Virus Replication Pregnant Not Pregnant Lung homogenate *p>0.120 days 3,5,7 3 dpi L: lung N: nasal cavity % Nucleoprotein Positive cells (Image Scope) N N Not Pregnant L Pregnant L 200 400 200 400

9. pg/ml (log 10 ) Days Post Infection Cellular Recruitment Factors Are Increased in Lungs of Pregnant Mice Luminex, Multiplex beads *p<0.05 Dotted line: mean value mock infected mice

10. pg/ml (log 10 ) Days Post Infection Th2 Cytokines Are Elevated in Pregnant Mice Pregnant Not Pregnant Luminex, Multiplex beads *p<0.05 Dotted line: mean value mock infected mice

11. Elevated Immune Cells in Lungs of Pregnant Mice *p<0.05 Dotted line: mock infected

12. Elevated Neutrophils in Alveolar Spaces & Interstitium Mock Pregnant Not Pregnant 220 Mock Virus

13. Elevated Macrophage Infiltration in Pregnant mice Mock Pregnant Not Pregnant 220 Mock Virus

14. Elevated Arginase -1 in Lungs of Pregnant mice Pregnant Not Pregnant Mock Virus 220

15. Mechanism of Action?  Macrophages and neutrophils can contribute to severe infections through the secretion of nitric oxide (NO)  NO has several functions:  Anti-viral signaling molecule  Vasodilator  vascular permeability  Impair tissue restoration Oda et al. 1989 Science; Kubes et al. 1992 Am J Physiol

16. Griess method Lung Repair is Impaired in Pregnant Mice *p<0.05 Pregnant Not Pregnant Naive Virus 400 *p<0.05 Arrows: attenuated epithelia NO responses in the Lung

17. Pregnant Mice Develop Increased Numbers of T Regulatory Cells in the Lung Gated on CD4 + CD25 + 9 dpi MLN: mediastinal lymph nodes

18. Summary Elevated Virus replication DCs MACS Lung Injury Tregs NO MACS Cytokines Th2>>Th1 MACS Neuts

19. http://www.searo.who.int/entity/emerging_diseases/topi cs/h1n1/en/index.html Therapies which modulate inflammation and lung repair processes may aid in reducing severity in pregnant women for future pandemics

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