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1 Depts. of Hematoly & Infectious Dis., 2 Div. of Hematopoiesis,

Early Phase Dynamics of HIV Infection in hPBMC-Transplanted NOD/SCID/Jak3 -/- Mice Using Infectious HIV Carrying a Fluorescent Protein, mCherry. Nobuyo Higashi-Kuwata 1 , H. Ogata-Aoki 1 , S. Hattori 2 , T. Nakamura 1 , M. Aoki 1 , S. Okada 2 , and H. Mitsuya 1,3.

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1 Depts. of Hematoly & Infectious Dis., 2 Div. of Hematopoiesis,

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  1. Early Phase Dynamics of HIV Infection in hPBMC-Transplanted NOD/SCID/Jak3-/- Mice Using Infectious HIV Carrying a Fluorescent Protein, mCherry Nobuyo Higashi-Kuwata1, H. Ogata-Aoki1, S. Hattori2, T. Nakamura1, M. Aoki1, S. Okada2, and H. Mitsuya1,3 1Depts. of Hematoly & Infectious Dis., 2Div. of Hematopoiesis, Center for AIDS Res., Kumamoto Univ., Japan, and 3Experimental Retrovirology Section, NCI, NIH, USA

  2. Background Certain early phase events in lenti-virus infection (e.g., HIV infection) have been examined, including the one by Ashley Haase and his colleagues. However, detailed dynamics and spreading profiles of HIV at early phases of initial infection yet remain to be determined. Experimental animal models that enable us to examine the effects of antiretroviral agents in the context of initial HIV transmission have not been developed. Haase AT et al. Nature464: 217-223 2010.

  3. Purpose The purpose of the present study is to examine and clarify the dynamics of HIV infection in hPBMC-transplanted NOD/SCID/ Jak3-/- (NOJ) mice using fluorescent protein, mCherry, which we hope may visualize the behavior of HIV. To this end, we first: 1. Generated HIV clones carrying a traceable fluorescent protein, mCherry. 2. Conditioned NOJ mice for establishing an in vivo model.

  4. Structure of HIV Containing the FP-encoding Gene (HIVmC) 7724 8761 8763 9413 gp41(1037bp) mCherry (708bp) nef (651bp) g gct pJRFL-based HIV clone Rev Vif Tat Gag LTR LTR Pol Env Nef mCherry Vpr Vpu

  5. Replication Rate of HIVmC is Comparable to That of HIVwt GMCSF MCSF 104 104 HIVwt HIVwt 103 103 p24 (ng/ml) HIVmC 102 102 HIVmC 101 101 14 14 0 9 0 9 Days after HIV-1inoculation GMCSF: Granulocyte Macrophage Colony Stimulating Factor MCSF: Macrophage Colony Stimulating Factor

  6. mCherry Signal is Detected as Early as Day 3 HIVwt HIVmC Day 3 Day 7 Day 11

  7. i.p. HIVmC Inoculation PROTOCOL Day -11 Day -10 Day 0 Day 7 Day 14 1.0 Gy X-ray Examine Examine i.p.2×107 hu-PBMC

  8. On Day 14, Human CD45+ Cells are Abundantly Seen in Various Organs Brain Liver Lung Spleen Skin Kidney Muscle Intestine Testis Vagina Lymph node

  9. By Day 14, HIVmC had Achieved High Levels of Replication p = 0.0012 107 106 p24 (pg/ml) 105 104 103 102 101 Day 7 Day 14

  10. i.p. HIVmC Inoculation PROTOCOL Day -11 Day -10 Day 0 Day 7 Day 14 1.0 Gy X-ray Examine Examine i.p.2 x 107 hu-PBMC Samples were subjected to stereoscopic imaging and immunohistochemistry

  11. Day 7: mCherry Signal is Detected Only in the Omentum mCherry Stereoscopic imaging (Maestro™) Stereomicroscopic imaging (Nuance™) HIV1-p24 1.0 mm 1.0 mm Composite mCherry In circulation: p24 antigen was undetectable. Composite mCherry mCherry Composite

  12. i.p. HIVmC Inoculation PROTOCOL Day -11 Day -10 Day 0 Day 7 Day 14 1.0 Gy X-ray Examine Examine i.p.2 x 107 hu-PBMC Samples were subjected to stereoscopic imaging and immunohistochemistry

  13. Day 14: mCherry Signal is Detected in Various Lymph Nodes Stereoscopic imaging (Maestro™) Stereomicroscopic imaging (Nuance™) Composite mCherry Gastric LNs Composite mCherry Mesenteric LNs mCherry Composite Iliac LNs Composite mCherry In circulation: p24 antigen = 18,400 pg/ml

  14. Day 14: mCherry+, p24+, hCD45+ Cells Found in Various Lymph Nodes HIV1-p24 mCherry hCD45 Gastric LNs Mesenteric LNs Iliac LNs

  15. Day 14: Lymph Nodes are Greater in Size & Number in HIV+ Mice than in HIV- Mice p = 0.054 p = 0.0005 0.30 10.0 0.20 Weight of lymph nodes (mg) 6.69 5.0 0.12 0.10 3.17 (not detected) 0 0 HIV- HIV- HIV+ HIV+ Celiac LNs Mesenteric LNs

  16. The Natural History of HIV-1 Transmission The Natural history of HIV-1 transmission is mediated by cell-associated and cell-free virions. We, therefore, compared the transmission efficiency by using cell-associated and cell-free virions.

  17. PROTOCOL Cell-free or Cell-free + Cell-associated Day -11 Day -10 Day 0 Day 7 Day 14 1.0 Gy X-ray Examine Examine i.p.2×107 hu-PBMC Plasma specimens were subjected to p24 determinations

  18. Cell-associated Virions Induce More Efficient & Faster Trans-mission than Cell-free Virions Day 7 Day 14 107 p = 0.008 n.s. 106 105 p24 (pg/ml) 104 103 102 101 Cell-free Cell-free Cell-associated Cell-associated

  19. Conclusions-1 Following intraperitoneal HIVmC inocu-lation, the virus penetrated through the visceral serosa and established infection in the lamina propria, followed by spreading through the vascular system throughout the body, generating productive infection. By day 7, mCherry signal was detected only in the omentum and by day 14, in the omentum and various lymph nodes.

  20. Conclusions-2 Lymph nodes were greater in size and number in HIVmC-innoculated mice than in uninfected mice. Transmission of HIVmC was more efficient with infection withcell-associated virionsthan withcell-free virions.

  21. Thank you for your attention

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