Biology subjects

Hope, T. J.

Publications and source records attributed to Hope, T. J..

At least 19 recordsLinked to original sources

bioRxiv2026

Senescence-associated loss of intestinal α1,2-fucose disrupts a modifiable host-microbiome homeostasis axis in people with HIV

BackgroundPeople with HIV (PWH), despite effective antiretroviral therapy (ART), experience disrupted intestinal homeostasis characterized by microbial dysbiosis and impaired intestinal barrier integrity, which contribute to chronic inflammation and aging-associated comorbidities. However, tractable mechanisms contributing to this dysfunction remain poorly defined. ObjectiveTo determine whether acquired loss of intestinal 1,2-fucose, a host-derived intrinsic prebiotic glycan that supports colonization by short-chain fatty acid (SCFA)-producing bacteria essential for intestinal barrier integrity, contributes to microbiome disruption, impaired epithelial resilience, inflammation, and biological aging in PWH. DesignIleal and colonic biopsies, isolated crypts, stool, and blood from PWH on ART and controls underwent multi-omic analyses. Findings were mechanistically interrogated using stool anaerobic fermentation assays and 3D intestinal organoid models of stress-mediated epithelial disruption. ResultsIn intestinal tissues, PWH exhibited reduced 1,2-fucosylation and increased senescence-associated expression of the fucose-degrading enzyme -L-fucosidase. Lower 1,2-fucose tracked with depletion of SCFA-producing bacteria, increased inflammation, and premature biological aging. In anaerobic fermentations, stool from PWH produced fewer SCFAs than controls, whereas supplementation with the human-milk-oligosaccharide-derived 1,2-fucose donor 2'-fucosyllactose restored SCFA production and improved intestinal organoid resilience to stress-mediated disruption. ConclusionThese findings identify acquired loss of intestinal 1,2-fucose as a modifiable host-microbiome mechanism linking epithelial senescence, microbial metabolic dysfunction, impaired barrier resilience, inflammation, and biological aging in treated HIV infection. SUMMARY BOXO_ST_ABSWhat is already known on this topicC_ST_ABSPeople with HIV on suppressive antiretroviral therapy frequently have persistent intestinal barrier dysfunction, microbial dysbiosis, chronic inflammation, and accelerated biological aging, but the host mechanisms that maintain this disrupted mucosal state remain incompletely defined. What this study addsThis study identifies acquired loss of intestinal 1,2-fucosylation as a feature of treated HIV infection and links this defect to a host fucosidase-high, senescence-enriched mucosal niche, depletion of SCFA-producing bacteria, impaired tight junction-associated barrier signatures, inflammation, and biological aging phenotypes. How this study might affect research, practice or policyThese findings support intestinal glycan ecology as a modifiable host-microbiome axis and provide a rationale for testing 1,2-fucose-replenishing strategies, such as 2'-fucosyllactose, to restore microbial metabolic output and improve epithelial resilience in people with HIV.

microbiology↗

bioRxiv2026

A Tissue Microenvironment Analogous to Certain Tumor Microenvironments Facilitates HIV Persistence

The HIV reservoir that establishes early upon infection and persists in tissues remains the primary barrier to a functional cure. While progress has been made to study the reservoir in blood compartments and specific cell types, knowledge gaps remain on the tissue microenvironment that facilitates persistence. The development of a novel immunoPET/CT-guided spatial transcriptomics pipeline has enabled the localization of foci of viral infection in tissues, rare events that are challenging to sample. Prior studies leveraging the pipeline have characterized the viral microenvironment (VME) gene signatures, cell types and interactions, and host transcriptome gene drivers of viral persistence. Detailed characterization of the VME revealed multiple shared features with certain tumor microenvironments (TMEs), suggesting shared immunoregulatory and survival mechanisms. In this study, we apply the immunoPET/CT-guided spatial transcriptomics pipeline in the SIVmac239/rhesus macaque model to define immune mechanisms underlying persistent versus transient HIV/SIV reservoirs. We utilize a systematic approach to highlight correlates between the VME and TME transcriptional programs, gene pathways, local tissue neighborhoods, cell-cell interactions, and host transcriptomic drivers. Analysis of broad transcriptional programs revealed SIV-localized spatial enrichment of genes associated with multiple cancer subtypes. The persistent reservoir was characterized by gene pathways of "cold" TMEs (e.g., epithelial to mesenchymal transition, TGF{beta} activation) whereas the transient reservoir was comparable to "hot" TMEs with cytotoxic immune activation. Cell-cell interaction analysis identified regulatory T cells as a key mediator of interactions in both persistent and transient reservoirs. Machine learning identified KRT8, EPCAM, and RRM2, genes with known roles in mediating carcinogenesis, among the top host transcriptomic drivers of the TME phenotype of the persistent VME. Collectively, the findings of this study provide novel and transformative insights on key mechanisms of HIV/SIV persistence and reveal potential targets for immunotherapeutic strategies aimed at reservoir disruption or clearance towards a functional HIV cure.

microbiology↗

bioRxiv2026

A Tissue Virus Microenvironment with Activated Stress Responses Underlies Durable SIV Persistence

HIV persistence during suppressive antiretroviral therapy (ART) remains a central barrier to cure, with the majority of reservoirs residing in gut-associated lymphoid tissues (GALT). Here, we define a spatially organized viral microenvironment (VME) that sustains reservoir durability and governs early viral rebound by comparing animals initiating ART early after infection (transient reservoirs) versus late (persistent reservoirs). Using immunoPET/CT-guided sampling of SIV-infected rhesus macaques combined with spatial transcriptomics, we interrogated tissue sites of viral production during the eclipse phase following analytical treatment interruption (ATI). Our results revealed that viral rebound from persistent reservoirs arises from discrete, transcriptionally active foci enriched in the mucosa lining the gut lumen. Eclipse phase persistent reservoirs were characterized by increased proviral burden and a distinct tissue state marked by activation of stress-response, metabolic, mitochondrial, and cell cycle programs coupled to repression of cytoplasmic translation and increased cellular senescence. These features co-occurred with immunosuppressive cellular architectures resembling tertiary lymphoid structures enriched for Treg cells, innate lymphoid cells, and mast cells, regulated by Treg-centered cell-cell interaction networks. In contrast, transient reservoirs displayed enhanced translational and metabolic activity and were embedded within immune-active environments enriched for CD8 T cells, Th17, Tfh, and activated CD4 T cells. Machine learning identified stress adaptation, hypoxia, metabolic rewiring, and cytoskeletal remodeling pathways as dominant predictors of viral density within persistent VMEs, with strong convergence on programs observed in tumor microenvironments (TME). Orthogonal validation confirmed activation of the integrated stress response (ISR) at sites of viral production in concurrence with results of immunofluorescent microscopy revealing SIV gag expression in two populations primarily in the mucosa, differentiated by the phosphorylation of eIF2. Together, these findings establish the VME as a critical determinant of reservoir persistence, integrating immune regulation, tissue remodeling, and translational control to enable viral survival. This framework suggests that effective HIV cure strategies will require coordinated disruption of VME-supportive functions in addition to targeting infected cells.

microbiology↗

bioRxiv2023

TGF-β blockade drives a transitional effector phenotype in T cells reversing SIV latency and decreasing SIV reservoirs in vivo

HIV-1 persistence during ART is due to the establishment of long-lived viral reservoirs in resting immune cells. Using an NHP model of barcoded SIVmac239 intravenous infection and therapeutic dosing of the anti-TGFBR1 inhibitor galunisertib (LY2157299), we confirmed the latency reversal properties of in vivo TGF-{beta} blockade, decreased viral reservoirs and stimulated immune responses. Eight SIV-infected macaques on ART were treated with four 2-week cycles of galunisertib. ART was discontinued 3 weeks after the last dose, and macaques euthanized 6 weeks after ART-interruption (ATI). 7 out of 8 macaques rebounded between week 2 and 6 post-ATI. Galunisertib led to viral reactivation as indicated by plasma viral load and immunoPET/CT with 64Cu-DOTA-F(ab)2-p7D3-probe. A decrease in cell-associated (CA-)SIV DNA was detected in lymph nodes, gut and PBMC, while intact pro-virus in PBMC decreased by 3-fold. No systemic increase in inflammatory cytokines was observed. High-dimensions cytometry, bulk, and single-cell (sc)RNAseq revealed a shift toward an effector phenotype in T and NK cells characterized by a progressive downregulation in TCF1. In summary, we demonstrated that galunisertib, a clinical stage TGF-{beta} inhibitor, reverses SIV latency and decreases SIV reservoirs by driving T cells toward an effector phenotype, enhancing immune responses in vivo in absence of toxicity. One-sentence summaryTGF-{beta} blockade drives an effector phenotype in immune cells leading to SIV latency reversal and enhanced immune responses in vivo.

immunology↗

bioRxiv2023

Temporal and spatial characterization of HIV/SIV infection at anorectal mucosa using rhesus macaque rectal challenge model

The study described herein is a continuation of our work in which we developed a methodology to identify small foci of transduced cells following rectal challenge of rhesus macaques with a non-replicative luciferase reporter virus. In the current study, the wild-type virus was added to the inoculation mix and twelve rhesus macaques were necropsied 2-4 days after the rectal challenge to study the changes in infected cell phenotype as the infection progressed. Relying on luciferase reporter we noted that both anus and rectum tissues are susceptible to the virus as early as 48h after the challenge. Small regions of the tissue containing luciferase-positive foci were further analyzed microscopically and were found to also contain cells infected by wild-type virus. Phenotypic analysis of the Env and Gag positive cells in these tissues revealed the virus can infect diverse cell populations, including but not limited to Th17 T cells, non Th17 T cells, immature dendritic cells, and myeloid-like cells. The proportions of the infected cell types, however, did not vary much during the first four days of infection when anus and rectum tissues were examined together. Nonetheless, when the same data was analyzed on a tissue-specific basis, we found significant changes in infected cell phenotypes over the course of infection. For anal tissue, a statistically significant increase in infection was observed for Th17 T cells and myeloid-like cells, while in the rectum, the non-Th17 T cells showed the biggest temporal increase, also of statistical significance. IMPORTANCEMen who have sex with men are at the highest risk of acquiring HIV via receptive anal intercourse. Understanding what sites are permissive to the virus, and what the early cellular targets are is critical for development of effective prevention strategies to control HIV acquisition amid receptive anal intercourse. Our work sheds light on the early HIV/SIV transmission events at the rectal mucosa by identifying the infected cells and highlights the distinct roles that different tissues play in virus acquisition and control.

immunology↗

bioRxiv2022

Contribution of 6mer seed toxicity to HIV-1 induced cytopathicity

HIV-1 (HIV) infects CD4 positive T cells, the gradual depletion of which can lead to the onset of Acquired Immunodeficiency Syndrome (AIDS) in the absence of antiretroviral therapy (ART). Several forms of cell death have been shown to be involved in HIV-mediated killing of either directly infected or bystander cells. It is still unknown, however, why some cells survive HIV infection and persist as part of the latently infected reservoir that reliably causes recurrent viremia upon ART cessation. Improved understanding of the mechanisms of HIV-mediated cell death could inform innovations designed to clear the latent reservoir. "Death Induced by Survival gene Elimination" (DISE) is an RNA interference (RNAi)-based mechanism that kills cells through short (s)RNAs with toxic 6mer seeds (pos. 2-7 of sRNA). These toxic seeds target reverse complementary seed matches in the 3UTR of mRNA transcripts to decrease expression of hundreds of genes that are critical for cell survival. In most cells under normal conditions, highly expressed cell-encoded non-toxic microRNAs (miRNAs) block access of toxic sRNAs to the RNA-induced silencing complex (RISC) that mediates RNAi, promoting cell survival. We now report that infection of cells with HIV results in RISC-loading of an HIV-encoded miRNA, v-miRNA HIV-miR-TAR-3p, which kills cells by DISE through a noncanonical (pos. 3-8) 6mer seed. In addition, cellular RISC bound sRNAs shift to lower seed viability. Both these effects also occur with latent HIV provirus reactivation in J-Lat cells, a well-established cell model of HIV latency. Cells lacking expression of miRNA biogenesis genes Drosha, Dicer and Exportin 5 cannot produce protective miRNAs and therefore do not block RISC loading of the v-miRNA HIV-miR-TAR-3p. These mutant cells, as well as cells lacking expression of the RISC component Ago2, are hypersensitive to cell death via DISE induced by HIV infection. More precise targeting of the balance between protective and cytotoxic sRNAs could specifically and transiently increase silencing of cell survival genes to increase DISE. This could be a new addition to a "shock and kill" strategy to enhance depletion of the provirus reservoir during suppressive ART.

microbiology↗

bioRxiv2022

Blockade of TGF-β signaling reactivates HIV-1/SIV reservoirs and immune responses in vivo

Elevated levels of TGF-{beta}, a potent immunosuppressive factor, are present in HIV-1 infected individuals even after years of antiretroviral therapy (ART). TGF-{beta} plays a critical role in maintaining immune cells in a resting state by inhibiting cell activation and proliferation. Resting HIV-1 target cells represent one of the main cellular reservoirs after long term ART and the low inducibility of the latent provirus constitutes one of the major obstacles to "kick and kill" cure strategies. We hypothesized that releasing cells from TGF-{beta}-driven signaling would promote latency reversal. To test our hypothesis, we compared ex vivo models of HIV-1 latency reactivation with and without TGF-{beta} and a TGF-{beta} type 1 receptor (TGFBR1) inhibitor, galunisertib. We also tested the effect of galunisertib in SIV infected, ART treated macaques by monitoring SIV envelope (env) protein expression via PET/CT using the Cu64-anti gp120 Fab (7D3) probe, along with plasma and tissue viral loads (VL). Exogenous TGF-1{beta} reduced HIV-1 reactivation in U1 and ACH2 latency models. Galunisertib increased HIV-1 latency reversal both in ex vivo models and in PBMC from HIV-1 infected, cART treated aviremic donors. In vivo, oral galunisertib promoted increased SIV env protein total standardized uptake values (SUVtot) in PET/CT images of tissues (gut and lymph nodes) of 5 out of 7 aviremic, long-term ART-treated, SIV-infected, macaques. This increase correlated with an increase in SIV RNA in gut tissue. Two out of 7 animals also exhibited increases in plasma viral load. Higher anti-SIV T cell responses and anti-SIV env antibody titers were detected after galunisertib treatment in most animals. In summary, our data suggest that blocking TGF-{beta} signaling simultaneously increases retroviral reactivation events and enhances anti-SIV immune responses.

immunology↗

bioRxiv2022

An immunoPET probe to SARS-CoV-2 reveals early infection of the male genital tract in rhesus macaques

The systemic nature of SARS-CoV-2 infection is highly recognized, but poorly characterized. A non-invasive and unbiased method is needed to clarify whole body spatiotemporal dynamics of SARS-CoV-2 infection after transmission. We recently developed a probe based on the anti-SARS-CoV-2 spike antibody CR3022 to study SARS-CoV-2 pathogenesis in vivo. Herein, we describe its use in immunoPET to investigate SARS-CoV-2 infection of three rhesus macaques. Using PET/CT imaging of macaques at different times post-SARS-CoV-2 inoculation, we track the 64Cu-labelled CR3022-F(ab)2 probe targeting the spike protein of SARS-CoV-2 to study the dynamics of infection within the respiratory tract and uncover novel sites of infection. Using this method, we uncovered differences in lung pathology between infection with the WA1 isolate and the delta variant, which were readily corroborated through computed tomography scans. The 64Cu-CR3022-probe also demonstrated dynamic changes occurring between 1- and 2-weeks post-infection. Remarkably, a robust signal was seen in the male genital tract (MGT) of all three animals studied. Infection of the MGT was validated by immunofluorescence imaging of infected cells in the testicular and penile tissue and severe pathology was observed in the testes of one animal at 2-weeks post-infection. The results presented here underscore the utility of using immunoPET to study the dynamics of SARS-CoV-2 infection to understand its pathogenicity and discover new anatomical sites of viral replication. We provide direct evidence for SARS-CoV-2 infection of the MGT in rhesus macaques revealing the possible pathologic outcomes of viral replication at these sites. Graphic AbstractPET/CT detected SARS-CoV-2 infection of 4 different tissues in the male genital tract illuminates the cause of COVID-19 clinical sequalae of male sexual health and fertility O_FIG O_LINKSMALLFIG WIDTH=189 HEIGHT=200 SRC="FIGDIR/small/481974v2_ufig1.gif" ALT="Figure 1"> View larger version (74K): org.highwire.dtl.DTLVardef@188c53aorg.highwire.dtl.DTLVardef@4c639forg.highwire.dtl.DTLVardef@120799forg.highwire.dtl.DTLVardef@110edb1_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO Diagram shows schematic illustration of the male genital tract of the rhesus macaque. Virus icon shows sites of SARS-CoV-2 PET signal. Text highlighting the clinical sequalae associated with each sight of infection is shown in text adjacent to each infection site. C_FIG

immunology↗

bioRxiv2022

Recognition of HIV-1 capsid licenses innate immune response to viral infection

Cyclic GMP-AMP synthase (cGAS) is a primary sensor of aberrant DNA that governs an innate immune signaling cascade, leading to the induction of the type-I interferon response. We have previously identified polyglutamine binding protein 1, PQBP1, as an adaptor molecule required for cGAS-mediated innate immune response of lentiviruses, including the human immunodeficiency virus 1 (HIV-1), but dispensable for the recognition of DNA viruses. HIV-1- encoded DNA is synthesized as a single copy from its RNA genome, and is subsequently integrated into the host chromatin. HIV-1 then produces progeny through amplification and packaging of its RNA genome, thus, in contrast to DNA viruses, HIV-1 DNA is both transient and of low abundance. However, the molecular basis for the detection and verification of this low abundance HIV-1 DNA pathogen-associated molecular pattern (PAMP) is not understood. Here, we elucidate a two-factor authentication strategy that is employed by the innate immune surveillance machinery to selectively respond to the low concentration of PAMP, while discerning these species from extranuclear DNA molecules. We find that, upon HIV-1 infection, PQBP1 decorates intact viral capsid, which serves as a primary verification step for the viral nucleic acid cargo. As the reverse transcription and capsid disassembly initiate, cGAS protein is then recruited to the capsid in a PQBP1-dependent manner, enabling cGAS molecules to be co-positioned at the site of PAMP generation. Thus, these data indicate that PQBP1 recognition of the HIV-1 capsid sanctions a robust cGAS-dependent response to a limited abundance and short-lived DNA PAMP. Critically, this illuminates a molecular strategy wherein the modular recruitment of co-factors to germline encoded pattern recognition receptors (PRRs) serves to enhance repertoire of pathogens that can be sensed by the innate immune surveillance machinery.

molecular biology↗

bioRxiv2021

Localization of infection in neonatal rhesus macaques after oral viral challenge

While vertical transmission of human immunodeficiency virus (HIV) can occur in utero and during delivery and through breastfeeding. We utilized Positron Emission Tomography (PET) imaging coupled with fluorescent microscopy of 64Cu-labeled photoactivatable-GFP-HIV (PA-GFP-BaL) to determine how HIV virions distribute and localize in neonatal rhesus macaques two and four hours after oral viral challenge. Our results show that by four hours after oral viral exposure, HIV virions localize to and penetrate the rectal mucosa. We also used a dual viral challenge with a non-replicative viral vector and a replication competent SHIV-1157ipd3N4 to examine viral transduction and dissemination at 96 hours. Our data show that while SHIV-1157ipd3N4 infection can be found in the oral cavity and upper gastrointestinal (GI) tract, the small and large intestine contained the largest number of infected cells. Moreover, we found that T cells were the biggest population of infected immune cells. Thus, thanks to these novel technologies, we are able to visualize and delineate of viral distribution and infection throughout the entire neonatal GI tract during acute viral infection. Author SummaryApproximately 1.8 million children are currently living with human immunodeficiency virus (HIV). While mother-to-child HIV transmission can occur in utero and during delivery, it most commonly occurs through breastfeeding, creating the need to understand how the virus moves throughout the body and infects the infant once breast milk is consumed. Here, we used multiple imaging techniques and PCR to determine how HIV distributes throughout the gastrointestinal tract after oral viral exposure and in which tissues and cell types become acutely infected. We found that HIV rapidly spreads throughout and penetrates the entire gastrointestinal tract as early as four hours after exposure. We also found that the intestine contained the largest number of infected cells at 96 hours and that most cells infected were T cells. Our study shows that these imaging technologies allow for the examination of viral distribution and infection in a rhesus macaque model.

cell biology↗

bioRxiv2021

Simultaneous in vivo Pharmacokinetics and Pharmacodynamics of TDF Intravaginal Ring during High-dose Vaginal SHIV Challenge in Pigtail Macaques

The demonstration of complete protection of macaques in a repeated low dose virus challenge by a tenofovir disoproxil fumarate (TDF) intravaginal ring (IVR) and the success of the dapivirine IVR in clinical trials highlighted the potential of IVRs as pre- exposure prophylaxis against HIV. Efficacy of TDF ring was not investigated in sexually active women. Our understanding of the mechanisms of protection is limited. To address this knowledge gap, we performed simultaneous pharmacokinetic and pharmacodynamic analysis of a TDF-IVR at the site of SIV challenge in pigtail macaques at the anatomical and cellular level. Specifically, we challenged TDF-IVR administered pigtail macaques with a single high dose of a non-replicative SIV-based vector containing a dual reporter system that helped us to identify the earliest targets of SIV infection within the mucosa. Two and three days after challenge, the macaques were euthanized and tenofovir (TFV) concentrations were measured in the female reproductive tract (FRT) by HPLC-MS/MS to correlate drug concentrations and SIV-vector transduction efficiency. TFV formed a gradient through the mucosal tissue, with the highest concentrations near the ring, in the upper vagina and endocervix. Despite this, several transduction events were identified with the most common sites being in the ovaries. Moreover, proviral DNA was detected in the cervix and vagina. Thus, our studies demonstrate an uneven distribution of TFV in the FRT of macaques after release from a TDF-IVR that leads to incomplete FRT protection from high viral dose challenge.

pharmacology and toxicology↗

bioRxiv2021

Effective prophylaxis of COVID-19 in rhesus macaques using a combination of two parentally-administered SARS-CoV-2 neutralizing antibodies

SARS-CoV-2 is a respiratory borne pathogenic beta coronavirus that is the source of a worldwide pandemic and the cause of multiple pathologies in man. The rhesus macaque model of COVID-19 was utilized to test the added benefit of combinatory parenteral administration of two high-affinity anti-SARS-CoV-2 monoclonal antibodies (mAbs; C144-LS and C135-LS) expressly developed to neutralize the virus and modified to extend their pharmacokinetics. After completion of kinetics study of mAbs in the primate, combination treatment was administered prophylactically to mucosal viral challenge. Results showed near complete virus neutralization evidenced by no measurable titer in mucosal tissue swabs, muting of cytokine/chemokine response, and lack of any discernable pathologic sequalae. Blocking infection was a dose-related effect, cohorts receiving lower doses (6, 2 mg/kg) resulted in low grade viral infection in various mucosal sites compared to that of a fully protective dose (20 mg/kg). A subset of animals within this cohort whose infectious challenge was delayed 75 days later after mAb administration were still protected from disease. Results indicate this combination mAb effectively blocks development of COVID-19 in the rhesus disease model and accelerates the prospect of clinical studies with this effective antibody combination.

microbiology↗

bioRxiv2021

Th17 T cells and immature dendritic cells are the preferential initial targets after rectal challenge with an SIV-based replication-defective dual-reporter vector

Understanding the earliest events of HIV sexual transmission is critical to develop and optimize HIV prevention strategies. To gain insights into the earliest steps of HIV rectal transmission, including cellular targets, rhesus macaques were intra-rectally challenged with a single-round SIV-based dual reporter that expresses luciferase and iRFP670 upon productive transduction. The vector was pseudotyped with the HIV-1 envelope JRFL. Regions of tissue containing foci of luminescent, transduced cells were identified macroscopically using an in vivo imaging system, and individual transduced cells expressing fluorescent protein were identified and phenotyped microscopically. This system revealed that anal and rectal tissues are both susceptible to transduction 48 hours after the rectal challenge. Detailed phenotypic analysis revealed that on average, 62% of transduced cells are CCR6+ T cells--the vast majority of which express ROR{gamma}T, a Th17 lineage-specific transcription factor. The second most common target cells were immature dendritic cells at 20%. These two cell types were transduced at the rates that are four to five times higher than their relative abundances indicate. Our work demonstrates that Th17 T and immature dendritic cells are preferential initial targets of HIV/SIV rectal transmission. IMPORTANCEMen and women who participate in unprotected receptive anal intercourse are at high risk for acquiring HIV. While in vitro data have developed a framework for understanding HIV cell tropism, the initial target cells in the rectal mucosa have not been identified. In this study, we identify these early host cells by using an innovative rhesus macaque rectal challenge model and methodology, which we previously developed. Thus, by shedding light on these early HIV/SIV transmission events, this study provides a specific cellular target for future prevention strategies.

immunology↗

bioRxiv2020

PET/CT targeted tissue sampling reveals virus specific dIgA can alter the distribution and localization of HIV after rectal exposure

HIV vaccines have not been successful in clinical trials. Dimeric IgA (dIgA) is the most abundant antibody class in mucosal tissues, making dIgA a prime candidate for potential HIV vaccines. We coupled Positron Emission Tomography (PET) imaging and fluorescent microscopy of 64Cu-labeled, photoactivatable-GFP HIV (PA-GFP-BaL) and fluorescently labeled dIgA to determine how dIgA antibodies influence virus interaction with mucosal barriers and viral penetration in colorectal tissue. Our results show that HIV virions rapidly disseminate throughout the colon two hours after exposure. The presence of dIgA resulted in an increase in virions and penetration depth in the transverse colon. Moreover, virions were found in the mesenteric lymph nodes two hours after viral exposure, and the presence of dIgA led to an increase in virions in mesenteric lymph nodes. Taken together, these technologies enable in vivo and in situ visualization of antibody-virus interactions and detailed investigations of early events in HIV infection.

cell biology↗

bioRxiv2020

Circulating ACE2-expressing Exosomes Block SARS-CoV-2 Virus Infection as an Innate Antiviral Mechanism

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the coronavirus disease 2019 (COVID-19) with innate and adaptive immune response triggered in such patients by viral antigens. Both convalescent plasma and engineered high affinity human monoclonal antibodies have shown therapeutic potential to treat COVID-19. Whether additional antiviral soluble factors exist in peripheral blood remain understudied. Herein, we detected circulating exosomes that express the SARS-CoV-2 viral entry receptor angiotensin-converting enzyme 2 (ACE2) in plasma of both healthy donors and convalescent COVID-19 patients. We demonstrated that exosomal ACE2 competes with cellular ACE2 for neutralization of SARS-CoV-2 infection. ACE2-expressing (ACE2+) exosomes blocked the binding of the viral spike (S) protein RBD to ACE2+ cells in a dose dependent manner, which was 400- to 700-fold more potent than that of vesicle-free recombinant human ACE2 extracellular domain protein (rhACE2). As a consequence, exosomal ACE2 prevented SARS-CoV-2 pseudotype virus tethering and infection of human host cells at a 50-150 fold higher efficacy than rhACE2. A similar antiviral activity of exosomal ACE2 was further demonstrated to block wild-type live SARS-CoV-2 infection. Of note, depletion of ACE2+ exosomes from COVID-19 patient plasma impaired the ability to block SARS-CoV-2 RBD binding to host cells. Our data demonstrate that ACE2+ exosomes can serve as a decoy therapeutic and a possible innate antiviral mechanism to block SARS-CoV-2 infection.

immunology↗

bioRxiv2019

Correlated cryogenic fluorescence microscopy and electron cryotomography shows that exogenous TRIM5α can form hexagonal lattices or autophagy aggregates in vivo

Members of the TRIM protein family have been shown to gather into structures in both the nucleus and cytoplasm. One TRIM protein family member, TRIM5, has been shown to form cytoplasmic bodies involved in restricting retroviruses such as HIV-1. Here we applied cryogenic correlated light and electron microscopy (cryo-CLEM) to intact mammalian cells expressing YFP-rhTRIM5 and found hexagonal nets were present whose arm-lengths were similar to those of the hexagonal nets formed by purified TRIM5 in-vitro. We also observed YFP-rhTRIM5 within a diversity of structures with characteristics expected for organelles involved in different stages of macroautophagy, including disorganized protein aggregations (sequestosomes), sequestosomes flanked by flat double-membraned vesicles (sequestosome:phagophore complexes), sequestosomes within a double-membraned vesicle (autophagosomes), and sequestosomes within multi-vesicular autophagic vacuoles (autolysosomes or amphisomes). Vaults were also seen in these structures, consistent with their role in autophagy. Our data (i) support recent reports that TRIM5 can form both well-organized signaling complexes and non-signaling aggregates, (ii) offer the first images of the macroautophagy pathway in a near-native state, and (iii) reveal that vaults arrive early in macroautophagy.

cell biology↗

bioRxiv2019

A Subcutaneous Implant of Tenofovir Alafenamide Fumarate Causes Local Inflammation and Tissue Necrosis in Rabbits and Macaques

We describe the in vitro and in vivo evaluation of a subcutaneous reservoir implant delivering tenofovir alafenamide hemifumarate (TAF) for the prevention of HIV infection. These long-acting reservoir implants were able to deliver antiretroviral drug for over 90 days in vitro and in vivo. We evaluated the implants for implantation site histopathology and pharmacokinetics in plasma and tissues for up to 12 weeks in New Zealand White rabbits and rhesus macaque models. A dose-ranging study in rabbits demonstrated dose-dependent pharmacokinetics and local inflammation up to severe necrosis around the active implants. The matched placebos showed normal wound healing and fibrous tissue encapsulation of the implant. We designed a second implant with a lower release rate and flux of TAF and achieved a median cellular level of tenofovir diphosphate of 42 fmol per 106 rhesus macaque peripheral blood mononuclear cells at a dose of 10 {micro}g/kg/day. This dose and flux of TAF also resulted in adverse local inflammation and necrosis near the implant in rhesus macaques. Inflammation in the primates was markedly lower in the placebo group than the active implant. The histological inflammatory response to the TAF implant at 4 and 12 weeks in primates was graded as a severe reaction. Thus, while we were able to achieve sustained target dose we observed unacceptable inflammatory response locally at the implant tissue interface.

pharmacology and toxicology↗

bioRxiv2019

Longitudinal bioluminescent imaging of HIV-1 infection during antiretroviral therapy and treatment interruption in humanized mice

Non-invasive bioluminescent imaging (NIBLI) of HIV-1 infection dynamics allows for real-time monitoring of viral spread and the localization of infected cell populations in living animals. In this report, we describe full-length replication-competent GFP and Nanoluciferase (Nluc) expressing HIV-1 reporter viruses from two clinical transmitted / founder (T/F) stains: TRJO.c and Q23.BG505. By infecting humanized mice with these HIV-1 T/F reporter viruses, we were able to directly monitor longitudinal viral spread at whole-animal resolution via NIBLI at a sensitivity of as few as 30-50 infected cells. Bioluminescent signal strongly correlated with HIV-1 infection and responded proportionally to virus suppression in vivo in animals treated daily with a combination antiretroviral therapy (cART) regimen. Longitudinal NIBLI following cART withdrawal visualized tissue-sites that harbored virus during infection recrudescence. Notably, we observed rebounding infection in the same lymphoid tissues where infection was first observed prior to ART treatment. Our work demonstrates the utility of our system for studying in vivo viral infection dynamics and identifying infected tissue regions for subsequent analyses.\n\nAuthor SummaryNon-invasive bioluminescent imaging (NIBLI) in small animals allows for in vivo longitudinal imaging of infection spread and pathogenesis. We have taken advantage of the small luciferase reporter protein, Nanoluciferase (Nluc), to generate a replication-competent HIV-1 reporter virus to allow for NIBLI of viral infection in humanize mice. NIBLI via Nluc enabled us to directly visualize longitudinal spreading patterns before, during, and after interruption of daily doses of combined antiretroviral therapy (cART). We observed that rebounding infection often emerged in tissue regions originally associated with infected cells prior to cART treatment. Thus, Nluc-based NIBLI of HIV-1 infection can be used as an experimental tool to study early events involved in viral dissemination and spread from initial sites of infection to draining lymphoid tissues as well as locate infected tissues for subsequent cellular characterization of HIV-1 infected cells.

microbiology↗