Mechanisms of Severity of Pandemic H1N Influenza A Virus Infection During Pregnancy, A Mouse Model Glendie Marcelin, PhD 3 rd World Congress on Virology November 21, 2013

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: Trudeau Institute

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

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

Lung Injury and alterations in immune responses responsible for severity?

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

*p= EID 50 pH1N1 is More Severe in Pregnant Mice

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

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

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

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

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

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

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

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 Science; Kubes et al Am J Physiol

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

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

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

cs/h1n1/en/index.html Therapies which modulate inflammation and lung repair processes may aid in reducing severity in pregnant women for future pandemics

Mission: NIAID funded bio-repository to provide reagents, tools and information for studying Category A, B, and C priority pathogens and emerging infectious disease agents, vectors of disease, non-pathogenic microbes relevant to human health and other microbiological materials of relevance to the research community. All materials are free of cost. Organism/Reagents: Viruses Antigens Assays/Panels Nucleic Acids Antibodies Antisera Peptide Arrays Bacteria Cell Banks Primers/Probes Parasitic Worms Rickettsiales Clones Fungi Protists Arthropod Vectors Toxins Libraries BEI Resources Collection and Reagents