Susceptibility to Rotavirus Infections. Mary K. Estes, PhD Baylor College of Medicine

Transcription

1 13 th International Rotavirus Symposium Session V: Advances in rotavirus immunology and virology Minsk, Belarus August 30, 2018 Susceptibility to Rotavirus Infections Mary K. Estes, PhD Baylor College of Medicine

2 Model Systems for Enteric Infections Small intestinal epithelium primary site of infection and replication for gastrointestinal viral pathogens Limited fundamental knowledge on intestinal epithelial cell-pathogen interactions Modeling interactions in vitro is difficult Cancer cell lines derived primarily from human colon Human rotaviruses replicate less efficiently in animal models New stem cell-derived nontransformed human intestinal enteroid (organoid) cultures are allowing new discoveries of host-virus interactions

3 Stem Cells Have Many Uses Development Therapy Regeneration Homeostasis Infectious Diseases Cancer We are using intestinal stem cells to understand gastrointestinal infections and human intestinal susceptibility and responses to infection 3

4 Human Intestinal Enteroid (HIE) Cultures Biopsy or Surgical tissue Isolate crypts Culture in media with growth factors Pioneered by Sato and Clevers (The Netherlands) Non-transformed, indefinite passaging All epithelial cell types enterocytes, goblet cells, enteroendocrine, Paneth cells Physiologically active Made from different intestinal segments Complexity and organization of the epithelium 3D / monolayer

5 Human Rotavirus Strains Infect Human Intestinal Enteroids Significantly Better Than Animal Strains Cell counts Uninfected Animal RV Human RV 0.5% RV+ 13% RV+ 50% RV+

6 Percent rotavirus positive Fold change 24/2 hpi infectious particles (FFU)/segment Human Rotavirus Infects Different Intestinal Segments Flow cytometry 300 Infectivity assay Duodenum Jejunum Ileum 0 Duodenum Jejunum Ileum Infection of enteroids from different patients and in different intestinal segments is reproducible

7 Rotavirus Infection in HIEs Rotavirus (red), E-cadherin (green) and nuclei (DAPI) Saxena et al., J Virol (2015) HIEs are physiologically responsive to rotavirus infections

8 Human Rotavirus Infects Enteroendocrine Cells and Induces Serotonin Secretion from HIEs S Serotonin e r o n in (nm) n M RV-Ito ChgA ChgA Ito Mock * * Ito RV-Ito M o c Mock ChgA Ito B h p i 1 2 h p i 2 4 h p i HRV infection leads to basal release of serotonin by 12 hpi Neurogenin-3 expressing HIEs Is serotonin production by infected enteroendocrine cells associated with vomiting? Are a subset of enteroendocrine cells infected?

9 Studying Genetic Susceptibility in HIEs Each HIE culture is an individual! Retains the genetic properties of that individual Allow comparisons across genetically diverse individuals Answer questions on heterogeneity of responses seen in field studies

10 Distinct Genetic Susceptibility of HIEs to Rotavirus and Norovirus Rotavirus Wa G1P[8] Norovirus GII.4 Sydney Susceptible Resistant Different from epi studies indicating secretor status association with severe rotavirus diarrhea

11 Attenuated Infectivity of Rotavirus Vaccines Infections in 6 jejunal HIEs 3 secretors, 3 non-secretors G1P[8] Wa strain, RV1 vaccine RV1 infection attenuated in secretor positive and secretor negative HIE cultures Infectivity in HIEs mimics heterogeneity seen in populations First in vitro culture system to show vaccine attenuation Saxena et al., J Virol (2015)

12 Transcriptional Response to Rotavirus (Across Segments) Infections with rotavirus and EAEC Duodenum, Ileum, Colon from the same person Segment-specific responses (same genetic background) Segment HIE Line % Infected Cells Duodenum D D Ileum IL IL Colon C C Jejunum J J

13 Common Pathways Upregulated! Reactome database analysis Top 3 pathways associated with upregulated genes HIEs show distinct transcriptional responses to viral versus bacterial infection

14 Human Intestinal Interferon Response to Rotavirus Dominant transcriptional pathway is type III IFN Endogenous response does not restrict virus replication In contrast, exogenous IFN treatment restricts virus replication Type I IFN more potent than type III IFN Extra-epithelial sources of type I IFN may be critical for limiting enteric virus replication Saxena K, PNAS 2017

15 Continued Development of HIE Cultures to Study Human-Virus-Microbe Interactions Mini Bioreactor Arrays for culture of microbiome ( R Britton, BCM) Bacteria Microbiome Parasites -crytosporidium Viruses enteroviruses, adenovirus Co-infections Vaccines and Innate Immunity HIE- macrophage cultures Future addition of nerves and vasculature Anaerobic culture conditions

16 Key Points New physiologically relevant tools (HIEs) can answer fundamental questions of public health importance Innate immune responses to rotavirus Genetic differences in HBGA expression and effects on susceptibility to vaccine viruses and other rotavirus strains Understand vaccine interference Coinfections with pathogens Test probiotics

17 A Great Basic Science and Clinical Team Khalil Ettayebi Sue Crawford Kosuke Murakami James Broughman Umesh Karandikar Victoria Tenge Fred Neill Sarah Blutt Shelly Zeng Kapil Saxena Baijun Kou Sashi Ramani Douglas Burrin Antone Opekun Robert Atmar David Graham

18 THANK YOU