Although effective antiretroviral therapy (ART) has substantially reduced the severity of human immunodeficiency virus (HIV)-associated neurocognitive disorders (HAND), the condition remains highly prevalent. Understanding HAND has been a challenge due to the lack of small animal models capable of supporting productive HIV infection in the brain. Recent advances in humanized mouse models with engrafted human glial cells now enable systemic HIV infection that extends to the central nervous system, offering a powerful platform to study HAND pathogenesis. In this study, we investigated behavioral alterations and neuropathological changes associated with HIV infection. Using home-cage monitoring, we observed that HIV-infected mice exhibited reduced feeding efficiency, consuming less food despite spending more time at the feeder, compared to uninfected controls. Additionally, infected animals displayed disrupted circadian rhythms, with a significant correlation between central nervous system viral load and increased locomotor activity during the light cycle. Neuropathological analyses revealed region-specific vulnerability, with the cortex exhibiting pronounced inflammatory and neurodegenerative changes. These findings were supported by transcriptomic profiling, which demonstrated heightened inflammatory and antiviral gene expression in the cortex, along with differentially expressed genes associated with neuropathology and behavioral deficits. Together, these results highlight distinct region-specific responses to HIV infection in the brain and establish this humanized mouse model as a valuable tool for elucidating the mechanisms underlying HAND and its associated behavioral deficits.

Liver disease is one of the leading comorbidities in HIV infection. The risk of liver fibrosis development is potentiated by alcohol abuse. In our previous studies, we reported that hepatocytes exposed to HIV and acetaldehyde undergo significant apoptosis, and the engulfment of apoptotic bodies (ABs) by hepatic stellate cells (HSC) potentiates their pro-fibrotic activation. However, in addition to hepatocytes, under the same conditions, ABs can be generated from liver-infiltrating immune cells. The goal of this study is to explore whether lymphocyte-derived ABs trigger HSC profibrotic activation as strongly as hepatocyte-derived ABs. ABs were generated from Huh7.5-CYP2E1 (RLW) cells and Jurkat cells treated with HIV+acetaldehyde and co-culture with HSC to induce their pro-fibrotic activation. ABs cargo was analyzed by proteomics. ABs generated from RLW, but not from Jurkat cells activated fibrogenic genes in HSC. This was driven by the expression of hepatocyte-specific proteins in ABs cargo. One of these proteins is Hepatocyte-Derived Growth Factor, for which suppression attenuates pro-fibrotic activation of HSC. In mice humanized with only immune cells but not human hepatocytes, infected with HIV and fed ethanol, liver fibrosis was not observed. We conclude that HIV+ABs of hepatocyte origin promote HSC activation, which potentially may lead to liver fibrosis progression.

Long-acting injectable (LAI) formulations promise to deliver patient benefits by overcoming issues associated with non-adherence. A preclinical assessment of semi-solid prodrug nanoparticle (SSPN) LAI formulations of emtricitabine (FTC) is reported here. Pharmacokinetics over 28 days were assessed in Wistar rats, New Zealand white rabbits, and Balb/C mice following intramuscular injection. Two lead formulations were assessed for the prevention of an HIV infection in NSG-cmah(-/-) humanised mice to ensure antiviral activities were as anticipated according to the pharmacokinetics. Cmax was reached by 12, 48, and 24 h in rats, rabbits, and mice, respectively. Plasma concentrations were below the limit of detection (2 ng/mL) by 21 days in rats and rabbits, and 28 days in mice. Mice treated with SSPN formulations demonstrated undetectable viral loads (700 copies/mL detection limit), and HIV RNA remained undetectable 28 days post-infection in plasma, spleen, lung, and liver. The in vivo data presented here demonstrate that the combined prodrug/SSPN approach can provide a dramatically extended pharmacokinetic half-life across multiple preclinical species. Species differences in renal clearance of FTC mean that longer exposures are likely to be achievable in humans than in preclinical models.

Recently, we found that both HIV and acetaldehyde, an alcohol metabolite, induce hepatocyte apoptosis, resulting in the release of large extracellular vesicles called apoptotic bodies (ABs). The engulfment of these hepatocyte ABs by hepatic stellate cells (HSC) leads to their profibrotic activation. This study aims to establish the mechanisms of HSC activation after engulfment of ABs from acetaldehyde and HIV-exposed hepatocytes (ABAGS+HIV). In vitro experiments were performed on Huh7.5-CYP (RLW) cells to generate hepatocyte ABs and LX2 cells were used as HSC. To generate ABs, RLW cells were pretreated for 24 h with acetaldehyde, then exposed overnight to HIV1ADA and to acetaldehyde for 96 h. Thereafter, ABs were isolated from cell suspension by a differential centrifugation method and incubated with LX2 cells (3:1 ratio) for profibrotic genes and protein analyses. We found that HSC internalized ABs via the tyrosine kinase receptor, Axl. While the HIV gag RNA/HIV proteins accumulated in ABs elicited no productive infection in LX2 and immune cells, they triggered ROS and IL6 generation, which, in turn, activated profibrotic genes via the JNK-ERK1/2 and JAK-STAT3 pathways. Similarly, ongoing profibrotic activation was observed in immunodeficient NSG mice fed ethanol and injected with HIV-derived RLW ABs. We conclude that HSC activation by hepatocyte ABAGS+HIV engulfment is mediated by ROS-dependent JNK-ERK1/2 and IL6 triggering of JAK-STAT3 pathways. This can partially explain the mechanisms of liver fibrosis development frequently observed among alcohol abusing PLWH.

Defining the latent human immunodeficiency virus type 1 (HIV-1) burden in the human brain during progressive infection is limited by sample access. Human hematopoietic stem cells (hu-HSCs)-reconstituted humanized mice provide an opportunity for this study. The model mimics, in measure, HIV-1 pathophysiology, transmission, treatment, and elimination in an infected human host. However, to date, brain HIV-1 latency in hu-HSC mice during suppressive antiretroviral therapy (ART) was not studied. To address this need, hu-HSC mice were administered long acting (LA) ART 14 days after HIV-1 infection was established. Animals were maintained under suppressive ART for 3 months, at which time HIV-1 infection was detected at low levels in brain tissue by droplet digital polymerase chain reaction (ddPCR) test on DNA. Notably, adoptive transfer of cells acquired from the hu-HSC mouse brains and placed into naive hu-HSC mice demonstrated viral recovery. These proof-of-concept results demonstrate replication-competent HIV-1 reservoir can be established in hu-HSC mouse brains that persists during long-term ART treatment. Hu-HSC mice-based mouse viral outgrowth assay (hu-MVOA) serves as a sensitive tool to interrogate latent HIV-1 brain reservoirs.

BACKGROUND AND AIMS:Approximately 3.5% of the global population is chronically infected with Hepatitis B Virus (HBV), which puts them at high risk of end-stage liver disease, with the risk of persistent infection potentiated by alcohol consumption. However, the mechanisms underlying the effects of alcohol on HBV persistence remain unclear. Here, we aimed to establish in vivo/ex vivo evidence that alcohol suppresses HBV peptides-major histocompatibility complex (MHC) class I antigen display on primary human hepatocytes (PHH), which diminishes the recognition and clearance of HBV-infected hepatocytes by cytotoxic T-lymphocytes (CTLs). METHODS:We used fumarylacetoacetate hydrolase (Fah)-/-, Rag2-/-, common cytokine receptor gamma chain knock-out (FRG-KO) humanized mice transplanted with human leukocyte antigen-A2 (HLA-A2)-positive hepatocytes. The mice were HBV-infected and fed control and alcohol diets. Isolated hepatocytes were exposed ex vivo to HBV 18-27-HLA-A2-restricted CTLs to quantify cytotoxicity. For mechanistic studies, we measured proteasome activities, unfolded protein response (UPR), and endoplasmic reticulum (ER) stress in hepatocytes from HBV-infected humanized mouse livers. RESULTS AND CONCLUSIONS:We found that alcohol feeding attenuated HBV core 18-27-HLA-A2 complex presentation on infected hepatocytes due to the suppression of proteasome function and ER stress induction, which diminished both the processing of HBV peptides and trafficking of HBV-MHC class I complexes to the hepatocyte surface. This alcohol-mediated decrease in MHC class I-restricted antigen presentation of the CTL epitope on target hepatocytes reduced the CTL-specific elimination of infected cells, potentially leading to HBV-infection persistence, which promotes end-stage liver disease outcomes.

Early-onset heart failure (HF) continues to be a major cause of morbidity and mortality in people living with human immunodeficiency virus type one (HIV-1) infection (PLWH), yet the molecular causes for this remain poorly understood. Herein NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ humanized mice (Hu-mice), plasma from PLWH, and autopsied cardiac tissues from deceased HIV seropositive individuals were used to assess if there is a link between the glycolysis byproduct methylglyoxal (MG) and HF in the setting of HIV-1 infection. At five weeks post HIV infection, Hu-mice developed grade III-IV diastolic dysfunction (DD) with an associated two-fold increase in plasma MG. At sixteen-seventeen weeks post infection, cardiac ejection fraction and fractional shortening also declined by 26 and 35%, and plasma MG increased to four-fold higher than uninfected controls. Histopathological and biochemical analyses of cardiac tissues from Hu-mice 17 weeks post-infection affirmed MG increase with a concomitant decrease in expression of the MG-degrading enzyme glyoxalase-1 (Glo1). The endothelial cell marker CD31 was found to be lower, and coronary microvascular leakage and myocardial fibrosis were prominent. Increasing expression of Glo1 in Hu-mice five weeks post-infection using a single dose of an engineered AAV2/9 (1.7 × 1012 virion particles/kg), attenuated the increases in plasma and cardiac MG levels. Increasing Glo1 also blunted microvascular leakage, fibrosis, and HF seen at sixteen weeks post-infection, without changes in plasma viral loads. In plasma from virally suppressed PLWH, MG was also 3.7-fold higher. In autopsied cardiac tissues from seropositive, HIV individuals with low viral log, MG was 4.2-fold higher and Glo1 was 50% lower compared to uninfected controls. These data show for the first time a causal link between accumulation of MG and HF in the setting of HIV infection.

Our current findings show that acetaldehyde accelerates endoplasmic reticulum (ER) stress by activating the unfolded protein response arms inositol-requiring enzyme 1α-X-box binding protein 1 and activation transcription factor (ATF)6α, but not phospho PKR-like ER kinase-phospho eukaryotic initiation factor 2α-ATF4-C/EBP homologous protein in hepatitis B virus (HBV)-transfected HepG2.2.15 cells. It also potentiates Golgi fragmentation, as evident by punctate distribution of Golgi proteins, GM130, trans-Golgi network 46, and Giantin. While concomitantly increasing HBV DNA and HBV surface antigen titers, acetaldehyde-induced ER stress suppresses the presentation of HBV peptide-major histocompatibility complex I complexes on hepatocyte surfaces, thereby promoting the persistence of HBV infection in the liver.

Nowadays, there is a strong request for the treatment of chronic HBV-infection with direct acting antivirals. Furthermore, prevalent human immunodeficiency virus (HIV-1) and hepatitis B (HBV) co-infections highlight an immediate need for dual long-acting and easily administered antivirals. To this end, we modified lamivudine (3TC), a nucleoside analog inhibitor of both viruses, into a lipophilic monophosphorylated prodrug (M23TC). Prior work demonstrated that nanoformulation of M23TC (NM23TC) enhanced drug stability, controlled dissolution and improved access to sites of viral replication. The present study evaluated the efficacy of a NM23TC in HBV-infected chimeric liver humanized mice. Levels of HBV DNA and HBsAg in plasma were monitored up to 8 weeks posttreatment. A single intramuscular dose of 75 mg/kg 3TC equivalents of nanoformulated NM23TC provided sustained drug levels and suppressed HBV replication in humanized mice for 4 weeks. The results support further development of this long-acting 3TC nanoformulation for HBV treatment and prevention.

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of their morbidity. The liver immunopathology caused by HIV-1 remains elusive. Small xenograft animal models with human hepatocytes and human immune system can recapitulate the human biology of the disease's pathogenesis. Herein, a protocol is described to establish a dual humanized mouse model through human hepatocytes and CD34+ hematopoietic stem/progenitor cells (HSPCs) transplantation, to study liver immunopathology as observed in HIV-infected patients. To achieve dual reconstitution, male TK-NOG (NOD.Cg-Prkdcscid Il2rgtm1Sug Tg(Alb-TK)7-2/ShiJic) mice are intraperitoneally injected with ganciclovir (GCV) doses to eliminate mouse transgenic liver cells, and with treosulfan for nonmyeloablative conditioning, both of which facilitate human hepatocyte (HEP) engraftment and human immune system (HIS) development. Human albumin (ALB) levels are evaluated for liver engraftment, and the presence of human immune cells in blood detected by flow cytometry confirms the establishment of human immune system. The model developed using the protocol described here resembles multiple components of liver damage from HIV-1 infection. Its establishment could prove to be essential for studies of hepatitis virus co-infection and for the evaluation of antiviral and antiretroviral drugs.

BACKGROUND:Microglia are the principal innate immune defense cells of the centeral nervous system (CNS) and the target of the human immunodeficiency virus type one (HIV-1). A complete understanding of human microglial biology and function requires the cell's presence in a brain microenvironment. Lack of relevant animal models thus far has also precluded studies of HIV-1 infection. Productive viral infection in brain occurs only in human myeloid linage microglia and perivascular macrophages and requires cells present throughout the brain. Once infected, however, microglia become immune competent serving as sources of cellular neurotoxic factors leading to disrupted brain homeostasis and neurodegeneration.METHODS:Herein, we created a humanized bone-marrow chimera producing human "microglia like" cells in NOD.Cg-PrkdcscidIl2rgtm1SugTg(CMV-IL34)1/Jic mice. Newborn mice were engrafted intrahepatically with umbilical cord blood derived CD34+ hematopoietic stem progenitor cells (HSPC). After 3 months of stable engraftment, animals were infected with HIV-1ADA, a myeloid-specific tropic viral isolate. Virologic, immune and brain immunohistology were performed on blood, peripheral lymphoid tissues, and brain.RESULTS:Human interleukin-34 under the control of the cytomegalovirus promoter inserted in NSG mouse strain drove brain reconstitution of HSPC derived peripheral macrophages into microglial-like cells. These human cells expressed canonical human microglial cell markers that included CD14, CD68, CD163, CD11b, ITGB2, CX3CR1, CSFR1, TREM2 and P2RY12. Prior restriction to HIV-1 infection in the rodent brain rested on an inability to reconstitute human microglia. Thus, the natural emergence of these cells from ingressed peripheral macrophages to the brain could allow, for the first time, the study of a CNS viral reservoir. To this end we monitored HIV-1 infection in a rodent brain. Viral RNA and HIV-1p24 antigens were readily observed in infected brain tissues. Deep RNA sequencing of these infected mice and differential expression analysis revealed human-specific molecular signatures representative of antiviral and neuroinflammatory responses.CONCLUSIONS:This humanized microglia mouse reflected human HIV-1 infection in its known principal reservoir and showed the development of disease-specific innate immune inflammatory and neurotoxic responses mirroring what can occur in an infected human brain.

In an era of improved survival due to modern antiretroviral therapy, liver disease has become a major cause of morbidity and mortality, resulting in death in 15–17% of human immunodeficiency virus (HIV)-infected patients. Alcohol enhances HIV-mediated liver damage and promotes the progression to advanced fibrosis and cirrhosis. However, the mechanisms behind these events are uncertain. Here, we hypothesize that ethanol metabolism potentiates accumulation of HIV in hepatocytes, causing oxidative stress and intensive apoptotic cell death. Engulfment of HIV-containing apoptotic hepatocytes by non-parenchymal cells (NPCs) triggers their activation and liver injury progression. This study was performed on primary human hepatocytes and Huh7.5-CYP cells infected with HIV-1ADA, and major findings were confirmed by pilot data obtained on ethanol-fed HIV-injected chimeric mice with humanized livers. We demonstrated that ethanol exposure potentiates HIV accumulation in hepatocytes by suppressing HIV degradation by lysosomes and proteasomes. This leads to increased oxidative stress and hepatocyte apoptosis. Exposure of HIV-infected apoptotic hepatocytes to NPCs activates the inflammasome in macrophages and pro-fibrotic genes in hepatic stellate cells. We conclude that while HIV and ethanol metabolism-triggered apoptosis clears up HIV-infected hepatocytes, continued generation of HIV-expressing apoptotic bodies may be detrimental for progression of liver inflammation and fibrosis due to constant activation of NPCs.

Hepatitis B virus (HBV) infection and alcoholism are major public health problems worldwide, contributing to the development of end-stage liver disease. Alcohol intake affects HBV infection pathogenesis and treatment outcomes. HBV-specific cytotoxic T lymphocytes (CTLs) play an important role in HBV clearance. Many previous studies have focused on alcohol-induced impairments of the immune response. However, it is not clear whether alcohol alters the presentation of HBV peptide-major histocompatibility complex (MHC) class I complexes on infected hepatocytes resulting in escape of its recognition by CTLs. Hence, the focus of this study was to investigate the mechanisms by which ethanol metabolism affects the presentation of CTL epitope on HBV-infected hepatocytes. As demonstrated here, although continuous cell exposure to acetaldehyde-generating system (AGS) increased HBV load in HepG2.2.15 cells, it decreased the expression of HBV core peptide 18-27-human leukocyte antigen-A2complex (CTL epitope) on the cell surface. Moreover, we observed AGS-induced suppression of chymotrypsin- and trypsin-like proteasome activities necessary for peptide processing by proteasome as well as a decline in IFNγ-stimulated immunoproteasome (IPR) function and expression of PA28 activator and immunoproteasome subunits LMP7 and LMP2. Furthermore, IFNγ-induced activation of peptide-loading complex (PLC) components, such as transporter associated with antigen processing (TAP1) and tapasin, were suppressed by AGS. The attenuation of IPR and PLC activation was attributed to AGS-triggered impairment of IFNγ signaling in HepG2.2.15 cells. Collectively, all these downstream events reduced the display of HBV peptide-MHC class I complexes on the hepatocyte surface, which may suppress CTL activation and the recognition of CTL epitopes on HBV-expressing hepatocytes by immune cells, thereby leading to persistence of liver inflammation.NEW & NOTEWORTHY Our study shows that in HBV-expressing HepG2.2.15 cells, acetaldehyde alters HBV peptide processing by suppressing chymotrypsin- and trypsin-like proteasome activities and decreases IFNγ-stimulated immunoproteasome function and expression of PA28 activator and immunoproteasome subunits. It also suppresses IFNγ-induced activation of peptide-loading complex (PLC) components due to impairment of IFNγ signaling via the JAK-STAT1 pathway. These acetaldehyde-induced dysfunctions reduced the display of HBV peptide-MHC class I complexes on the hepatocyte surface, thereby leading to persistence of HBV infection.