第1319回生物科学セミナー
From PET/CT to electron microscopy: Utilizing Multiscale Imaging to Bring Anatomy and Physiology to HIV Prevention and Cure Research.
Thomas J Hope, PhD（Northwestern University, Feinberg School of Medicine, Chicago, IL.）
2020年02月28日(金) 16:00-17:00 理学部1号館東棟 380号室
Different imaging modalities can provide unique insights into the localization of labelled proteins and factors at different scales. For example, positronic emission tomography (PET) allows the detection of radiolabeled factors deep within tissue, with resolution at the millimeter scale. Likewise, bioluminescence also provides similar millimeter resolution, however photons are much less efficient at passing through tissue limiting its use in large animals models. Fluorescent light-sheet imaging facilitates the evaluation of fluorescently tagged components at a resolution at 10-100 µM level, but requires tissue clearing to minimize photon dispersion and increase resolution. These approaches providing insights at the whole animal and tissue level can be correlated with different fluorescent microscopy and fluorescent super resolution approaches, along with electron microscopy approaches to allow imaging and insights over 9 orders of magnitude from meters to nanometers. We have recently refined the ability to correlate these multiscale imaging approaches to provide insights into SIV/SHIV/HIV transmission, dissemination, reservoirs, and prevention in the rhesus macaque model.
Approaches correlating PET and/or bioluminescence provide a novel and unbiased look at the localization of virus, antibodies, and infected/transduced cells within the context of tissue and living animals. For example, as we have previously reported, bioluminescence generated by firefly luciferase delivered by a dual reporter vector revealed that the upper female reproductive tract including the uterus and ovaries, is highly susceptible to SIV infection after vaginal challenge in macaque models (Steih et al 2014, PLoS Pathogens, Stieh et al 2016, CHM). PET scans can be utilized in a similar way and are superior to bioluminescence in macaque models. PET scans allow the efficient visualization of radioactive signal in the context of a living animal at multiple time points providing novel kinetic insights into anatomical distribution and half-life. By combining fluorescent labeling with radioactive labeling it is possible to validate that the radioactive signal represents the distribution of the intact labeled protein or viral particle rather than a metabolic product of the radiolabeled modality. Studies with 64Cu-labeled antibodies have revealed that the tissue distribution of antibodies after iv injection is asymmetric, heterogenous, and requires approximately 1 week to achieve steady state tissue distribution. Rectal instillation of 64Cu-labeled viral particles reveals that the particles accumulate beyond the rectum to reach the entire descending colon, and even aspects of the transverse colon. Unexpectedly, viral particles can reach a subset of draining mesenteric lymph nodes (LN) within 2 hours of rectal instillation. Utilizing our photoactivatable virus technology (Carias et al, 2013, Journal of Virology) we can validate that intact virions, are present within the mesenteric LNs. Additionally, rectal instillation of virus specific dIgA1/2 can enhance viral particle distribution within colon and increase virus penetration within the mucosa. Virus specific dIgA2 can enhance virus distribution to the draining mesenteric LNs. The dIgA1/2-virus immune complexes are likely passed through the mucosal barrier by endogenous mucosal antigen sampling pathways and and carried to the draining mesenteric lymph nodes (LN) by dendritic cells as best defined in mouse mucosal immunology. Finally, using PET based detection of infected cells and virus by 64Cu-labeled SIV envelope specific Fab2 fragments reveals dramatic dissemination in the gut, LNs, and spleen after initiation of potent suppressive ART 4 days post vaginal and rectal challenge. Under these conditions, no virus is detected in the blood even though the envelope specific Fab2 signal is widely disseminated within tissues. This signal slowly decreases over weeks of treatment, persisting at detectable levels for months. It is clear that this correlative PET approach is revealing the fields naivety relating to the transmission, dissemination, and viral reservoirs in the context of the anatomy and physiology of a living macaque and analogously humans.
