Hepatitis B virus has infected a third of the world’s population, and 296 million people are living with chronic infection. Chronic infection leads to progressive liver disease, including hepatocellular carcinoma and liver failure, and there remains no reliable curative therapy. These gaps in our understanding are due, in large part, to a paucity of animal models of HBV infection. Here, we show that rhesus macaques regularly clear acute HBV infection, similar to adult humans, but can develop long-term infection if immunosuppressed. Similar to patients, we longitudinally detected HBV DNA, HBV surface antigen, and HBV e antigen in the serum of experimentally infected animals. In addition, we discovered hallmarks of HBV infection in the liver, including RNA transcription, HBV core and HBV surface antigen translation, and covalently closed circular DNA biogenesis. This pre-clinical animal model will serve to accelerate emerging HBV curative therapies into the clinic.
CRISPR/Cas systems are some of the most promising tools for therapeutic genome editing. The use of these systems is contingent on the optimal designs of guides and homology-directed repair (HDR) templates. While this design can be achieved in silico, validation and further optimization are usually performed with the help of reporter systems. Here, we describe a novel reporter system, termed BETLE, that allows for the fast, sensitive, and cell-specific detection of genome editing and template-specific HDR by encoding multiple reporter proteins in different open-reading frames. Out-of-frame non-homologous end joining (NHEJ) leads to the expression of either secretable NanoLuc luciferase, enabling a highly sensitive and low-cost analysis of editing, or fluorescent mTagBFP2, allowing for the enumeration and tissue-specific localization of genome-edited cells. BETLE includes a site to validate CRISPR/Cas systems for a sequence-of-interest, making it broadly adaptable. We evaluated BETLE using a defective moxGFP with a 39-base-pair deletion and showed spCas9, saCas9, and asCas12a editing as well as sequence-specific HDR and the repair of moxGFP in cell lines with single and multiple reporter integrants. Taken together, these data show that BETLE allows for the rapid detection and optimization of CRISPR/Cas genome editing and HDR in vitro and represents a state-of the art tool for future applications in vivo.
Few countermeasures to treat Yellow Fever virus (YFV) infection are under development, because vaccines have helped to limit new infections. Unfortunately, vaccine hesitancy, supply deficits, and a paucity of therapeutic options have left individuals at risk. Here, we tested potent YFV-specific neutralizing monoclonal antibodies in rodents and non-human primates. We administered antibodies during acute pathogenic YFV infection and demonstrate that we can prevent severe disease and death. Given the severity of YFV-induced disease, our results show that these antibodies could be effective in saving lives and fill a much-needed void in managing Yellow Fever cases during outbreaks around the world.
Non-alcoholic fatty liver disease (NAFLD) contributes to morbidity and mortality in HIV-infected individuals. To evaluate liver immune changes during SIV infection of macaques, we obtained liver biopsies laparoscopically at 2, 6, and 16 weeks post-infection as well as at necropsy (32 weeks). SIV infection was associated with liver dysfunction that included elevated liver enzymes during acute infection, higher liver weight at necropsy, and histologic changes within the liver lobule (functional unit of the liver) including sinusoidal dilatation near central veins and portal expansion. The type-1 interferon (IFN-1) response (MX1 expression) increased over time post-SIV infection, initially being observed in macrophages (week 2) with a switch to a mixed hepatocyte/monocyte/macrophage response by 16–32 weeks. Over the course of SIV infection, macrophage frequencies also increased – both infiltrating (CD68+CD163+CD206-) and resident (CD68+CD163+CD206+) tissue macrophages – and these macrophages were found more near central veins over time. In contrast, CD68+CD163-CD206− trafficking myeloid cells/monocytes were detected predominantly in the periportal zone of the lobule and associated with elevated MX1 expression in that zone. Our findings identify pathologic changes within the livers of SIV infected macaques as early as 2 weeks post-infection, IFN-1 responses that are initially observed in liver macrophages, and an accumulation of macrophages associated with signs of inflammation by week 32. These studies offer quantitative and spatial insights to inform macrophage-targeted therapeutic approaches that may improve liver function during HIV infection. Supported by grants from NIH (R01AI134630)
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