TGF-ß 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 anti-TGFBR1 inhibitor galunisertib (LY2157299), we confirm the latency reversal properties of in vivo TGF-ß blockade, decrease viral reservoirs and stimulate immune responses. Treatment of eight female, SIV-infected macaques on ART with four 2-weeks cycles of galunisertib leads to viral reactivation as indicated by plasma viral load and immunoPET/CT with a 64Cu-DOTA-F(ab')2-p7D3-probe. Post-galunisertib, lymph nodes, gut and PBMC exhibit lower cell-associated (CA-)SIV DNA and lower intact pro-virus (PBMC). Galunisertib does not lead to systemic increase in inflammatory cytokines. High-dimensional cytometry, bulk, and single-cell (sc)RNAseq reveal a galunisertib-driven shift toward an effector phenotype in T and NK cells characterized by a progressive downregulation in TCF1. In summary, we demonstrate that galunisertib, a clinical stage TGF-ß 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.

TGF-ß 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-ß blockade, decreased viral reservoirs and stimulated immune responses. Eight SIV-infected macaques on suppressive ART were treated with 4 2-week cycles of galunisertib. ART was discontinued 3 weeks after the last dose, and macaques euthanized 6 weeks after ART-interruption(ATI). One macaque did not rebound, while the remaining rebounded between week 2 and 6 post-ATI. Galunisertib led to viral reactivation as indicated by plasma viral load and immunoPET/CT with the 64Cu-DOTA-F(ab')2-p7D3-probe. Half to 1 Log 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 RNAseq revealed a shift toward an effector phenotype in T and NK cells. In summary, we demonstrated that galunisertib, a clinical stage TGFß 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.

Species-agnostic polymeric formulations for inhalable messenger RNA delivery to the lung.

Messenger RNA has now been used to vaccinate millions of people. However, the diversity of pulmonary pathologies, including infections, genetic disorders, asthma and others, reveals the lung as an important organ to directly target for future RNA therapeutics and preventatives. Here we report the screening of 166 polymeric nanoparticle formulations for functional delivery to the lungs, obtained from a combinatorial synthesis approach combined with a low-dead-volume nose-only inhalation system for mice. We identify P76, a poly-ß-amino-thio-ester polymer, that exhibits increased expression over formulations lacking the thiol component, delivery to different animal species with varying RNA cargos and low toxicity. P76 allows for dose sparing when delivering an mRNA-expressed Cas13a-mediated treatment in a SARS-CoV-2 challenge model, resulting in similar efficacy to a 20-fold higher dose of a neutralizing antibody. Overall, the combinatorial synthesis approach allowed for the discovery of promising polymeric formulations for future RNA pharmaceutical development for the lungs.

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

TGF-ß plays a critical role in maintaining immune cells in a resting state by inhibiting cell activation and proliferation. Resting HIV-1 target cells represent the main cellular reservoir after long-term antiretroviral therapy (ART). We hypothesized that releasing cells from TGF-ß-driven signaling would promote latency reversal. To test our hypothesis, we compared HIV-1 latency models with and without TGF-ß and a TGF-ß type 1 receptor inhibitor, galunisertib. We tested the effect of galunisertib in SIV-infected, ART-treated macaques by monitoring SIV-env expression via PET/CT using the 64Cu-DOTA-F(ab')2 p7D3 probe, along with plasma and tissue viral loads (VLs). Exogenous TGF-ß reduced HIV-1 reactivation in U1 and ACH-2 models. Galunisertib increased HIV-1 latency reversal ex vivo and in PBMCs from HIV-1-infected, ART-treated, aviremic donors. In vivo, oral galunisertib promoted increased total standardized uptake values in PET/CT images in 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 the gut. Two of the 7 animals also exhibited increases in plasma VLs. Higher anti-SIV T cell responses and antibody titers were detected after galunisertib treatment. In summary, our data suggest that blocking TGF-ß signaling simultaneously increases retroviral reactivation events and enhances anti-SIV immune responses.

Localization of infection in neonatal rhesus macaques after oral viral challenge.

Vertical transmission of human immunodeficiency virus (HIV) can occur in utero, 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.

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

Human immunodeficiency virus (HIV) vaccines have not been successful in clinical trials. Dimeric IgA (dIgA) in the form of secretory IgA 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.

Anatomic Distribution of Intravenously Injected IgG Takes Approximately 1 Week to Achieve Stratum Corneum Saturation in Vaginal Tissues.

i.v. injected Abs have demonstrated protection against simian HIV infection in rhesus macaques, paving the way for the Antibody Mediated Prevention trial in which at-risk individuals for HIV received an i.v. infusion of the HIV broadly neutralizing Ab VRC01. However, the time needed for these Abs to fully distribute and elicit protection at mucosal sites is still unknown. In this study, we interrogate how long it takes for Abs to achieve peak anatomical levels at the vaginal surface following i.v. injection. Fluorescently labeled VRC01 and/or Gamunex-C were i.v. injected into 24 female rhesus macaques (Macaca mulatta) with vaginal tissues and plasma acquired up to 2 wk postinjection. We found that Ab delivery to the vaginal mucosa occurs in two phases. The first phase involves delivery to the submucosa, occurring within 24 h and persisting beyond 1 wk. The second phase is the delivery through the stratified squamous epithelium, needing ∼1 wk to saturate the stratum corneum. This study has important implications for the efficacy of immunoprophylaxis targeting pathogens at the mucosa.

Comparison of predictors for terminal disease progression in simian immunodeficiency virus/simian-HIV-infected rhesus macaques.

OBJECTIVES: CD4+ T-cell decline and increasing virus levels are considered hallmarks of HIV/AIDS pathogenesis but we previously demonstrated in rhesus macaques that tissue macrophage destruction by simian immunodeficiency virus (SIV) infection associated with increased monocyte turnover also appear to impact pathogenesis. It remains unclear, however, which factors best predict onset of terminal disease progression and survival time. The objective of this study, therefore, was to directly compare these co-variates of infection for predicting survival times in retrospective studies of SIV/simian-HIV (SHIV)-infected adult rhesus macaques. METHODS: Rhesus macaques were infected with various strains of SIV/SHIV and evaluated longitudinally for monocyte turnover, CD4+ T-cell loss, plasma viral load, and SIV/SHIV strain. Correlation analyses and machine learning algorithm modeling were applied to compare relative contributions of each of the co-variates to survival time. RESULTS: All animals with AIDS-related clinical signs requiring euthanasia exhibited increased monocyte turnover regardless of CD4+ T-cell level, viral strain, or plasma viral load. Regression analyses and machine learning algorithms indicated a stronger correlation and contribution between increased monocyte turnover and reduced survival time than between CD4+ T-cell decline, plasma viral load, or virus strain and reduced survival time. Decision tree modeling categorized monocyte turnover of 13.2% as the initial significant threshold that best predicted decreased survival time. CONCLUSION: These results demonstrate that monocytes/macrophages significantly affect HIV/SIV pathogenesis outcomes. Monocyte turnover analyses are not currently feasible in humans, so there is a need to identify surrogate biomarkers reflecting tissue macrophage damage that predict HIV infection disease progression.

Simian Immunodeficiency Virus Susceptibility, Immunology, and Microbiome in the Female Genital Tract of Adolescent Versus Adult Pigtail Macaques.

In Sub-Saharan Africa, young women 15-24 years of age account for nearly 30% of all new HIV infections, however, biological and epidemiological factors underlying this disproportionate infection rate are unclear. In this study, we assessed biological contributors of SIV/HIV susceptibility in the female genital tract (FGT) using adolescent (n = 9) and adult (n = 10) pigtail macaques (PTMs) with weekly low-dose intravaginal challenges of SIV. Immunological variables were captured in vaginal tissue of PTMs by flow cytometry and cytokine assays. Vaginal biopsies were profiled by proteomic analysis. The vaginal microbiome was assessed by 16S rRNA sequencing. We were powered to detect a 2.2-fold increase in infection rates between age groups, however, we identified no significant differences in susceptibility. This model cannot capture epidemiological factors or may not best represent biological differences of HIV susceptibility. No immune cell subsets measured were significantly different between groups. Inflammatory marker MCP-1 was significantly higher (adj p = .02), and sCD40L trended higher (adj p = .06) in vaginal cytobrushes of adults. Proteomic analysis of vaginal biopsies showed no significant (adj p < .05) protein or pathway differences between groups. Vaginal microbiomes were not significantly different between groups. No differences were observed between age groups in this PTM model, however, these animals may not reflect biological factors contributing to HIV risk such as those found in their human counterparts. This model is therefore not appropriate to explore human adolescent differences in HIV risk. Young women remain a key population at risk for HIV infection, and there is still a need for comprehensive assessment and intervention strategies for epidemic control of this uniquely vulnerable population.

Aerosol Delivery of Synthetic mRNA to Vaginal Mucosa Leads to Durable Expression of Broadly Neutralizing Antibodies against HIV.

There is a clear need for low-cost, self-applied, long-lasting approaches to prevent human immunodeficiency virus (HIV) infection in both men and women, even with the advent of pre-exposure prophylaxis (PrEP). Broadly neutralizing antibodies represent an option to improve HIV prophylaxis, but intravenous delivery, cold-chain stability requirements, low cervicovaginal concentrations, and cost may preclude their use. Here, we present an approach to express the anti-GP120 broadly neutralizing antibody PGT121 in the primary site of inoculation, the female reproductive tract, using synthetic mRNA. Expression is achieved through aerosol delivery of unformulated mRNA in water. We demonstrated high levels of antibody expression for over 28 days with a single mRNA administration in the reproductive tract of sheep. In rhesus macaques, neutralizing antibody titers in secretions developed within 4 h and simian-HIV (SHIV) infection of ex vivo explants was prevented. Persistence of PGT121 in vaginal secretions and epithelium was achieved through the incorporation of a glycosylphosphatidylinositol (GPI) anchor into the heavy chain of the antibody. Overall, we present a new paradigm to deliver neutralizing antibodies to the female reproductive tract for the prevention of HIV infections.

Visualization of early events in mRNA vaccine delivery in non-human primates via PET-CT and near-infrared imaging.

Visualization of the spatio-temporal trafficking of vaccines after their delivery would help evaluate the efficacy of candidate formulations and aid their rational design for preclinical and translational studies. Here, we show that a dual radionuclide-near-infrared probe allows for quantitative, longitudinal and non-invasive monitoring, via positron emission tomography-computed tomography and near-infrared imaging of cynomolgus macaques, of the trafficking dynamics to draining lymph nodes of a model messenger RNA vaccine labelled with the probe. After intramuscular administration of the vaccine to the monkeys, we observed the dynamics of the mRNA vaccine at the injection site and in the draining lymph nodes, performed cellular analyses of the involved tissues using flow cytometry and identified through immunofluorescence that professional antigen-presenting cells are the primary cells containing the injected mRNA and encoding the antigen. This approach may reveal spatio-temporal determinants of vaccine efficacy in preclinical and translational studies employing large mammals.

Optimizing the preparation and stability of decorated antiretroviral drug nanocrystals.

AIM: While the  therapeutic potential for current long-acting (LA) antiretroviral therapy (ART) is undeniable, ligand-decorated nanoformulated LA-ART could optimize drug delivery to viral reservoirs. The development of decorated ART hinges, however, on formulation processes and manufacture efficiencies. To this end, we compared manufacture and purification techniques for ligand-decorated antiretroviral drug nanocrystals. MATERIALS & METHODS: Ligand-decorated nanoparticle manufacturing was tested using folic acid (FA) nanoformulated cabotegravir. RESULTS: Direct manufacturing of FA-cabotegravir resulted in stable particles with high drug loading and monocyte-macrophage targeting. A one step 'direct' scheme proved superior over differential centrifugation or tangential flow filtration facilitating particle stability and preparation simplicity and efficiency. CONCLUSION: Direct manufacturing of FA nanoparticles provides a path toward large-scale clinical grade manufacturing of cell-targeted LA-ART.

High Turnover of Tissue Macrophages Contributes to Tuberculosis Reactivation in Simian Immunodeficiency Virus-Infected Rhesus Macaques.

Background: Tuberculosis (TB) and human immunodeficiency virus (HIV)/acquired immune deficiency syndrome (AIDS) profoundly affect the immune system and synergistically accelerate disease progression. It is believed that CD4+ T-cell depletion by HIV is the major cause of immunodeficiency and reactivation of latent TB. Previous studies demonstrated that blood monocyte turnover concurrent with tissue macrophage death from virus infection better predicted AIDS onset than CD4+ T-cell depletion in macaques infected with simian immunodeficiency virus (SIV). Methods: In this study, we describe the contribution of macrophages to the pathogenesis of Mycobacterium tuberculosis (Mtb)/SIV coinfection in a rhesus macaque model using in vivo BrdU labeling, immunostaining, flow cytometry, and confocal microscopy. Results: We found that increased monocyte and macrophage turnover and levels of SIV-infected lung macrophages correlated with TB reactivation. All Mtb/SIV-coinfected monkeys exhibited declines in CD4+ T cells regardless of reactivation or latency outcomes, negating lower CD4+ T-cell levels as a primary cause of Mtb reactivation. Conclusions: Results suggest that SIV-related damage to macrophages contributes to Mtb reactivation during coinfection. This also supports strategies to target lung macrophages for the treatment of TB.

Proteome analysis of bronchoalveolar lavage from calves infected with bovine respiratory syncytial virus-Insights in pathogenesis and perspectives for new treatments.

Human and bovine respiratory syncytial viruses (HRSV/BRSV) are major causes of severe lower respiratory tract infections in children and calves, respectively. Shared epidemiological, clinical, pathological and genetic characteristics of these viruses make comparative research highly relevant. To characterise the host response against BRSV infection, bronchoalveolar lavage supernatant (BAL) from i) non-vaccinated, BRSV-infected ii) vaccinated, BRSV-infected and iii) non-infected calves was analysed by tandem mass spectrometry. Proteins were semi-quantified and protein expression was validated by immunoblotting. Correlations between selected proteins and pathology, clinical signs and virus shedding were investigated. Calves with BRSV-induced disease had increased total protein concentrations and a decreased number of proteins identified in BAL. The protein profile was characterised by neutrophil activation and a reduction in identified antioxidant enzymes. The presence of neutrophils in alveolar septa, the expression level of neutrophil-related or antioxidant proteins and LZTFL1 correlated significantly with disease. Citrullinated histone 3, an indicator of extracellular traps (ETs), was only detected in non-vaccinated, BRSV-infected animals. By bringing disequilibrium in the release and detoxification of reactive oxygen species, generating ETs and causing elastine degradation, exaggerated neutrophil responses might exacerbate RSV-induced disease. Neutrophil-mitigating or antioxidant treatments should be further explored.

A mature macrophage is a principal HIV-1 cellular reservoir in humanized mice after treatment with long acting antiretroviral therapy.

BACKGROUND: Despite improved clinical outcomes seen following antiretroviral therapy (ART), resting CD4+ T cells continue to harbor latent human immunodeficiency virus type one (HIV-1). However, such cells are not likely the solitary viral reservoir and as such defining where and how others harbor virus is imperative for eradication measures. To such ends, we used HIV-1ADA-infected NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ mice reconstituted with a human immune system to explore two long-acting ART regimens investigating their abilities to affect viral cell infection and latency. At 6 weeks of infection animals were divided into four groups. One received long-acting (LA) cabotegravir (CAB) and rilpivirine (RVP) (2ART), a second received LA CAB, lamivudine, abacavir and RVP (4ART), a third were left untreated and a fourth served as an uninfected control. After 4 weeks of LA ART treatment, blood, spleen and bone marrow (BM) cells were collected then phenotypically characterized. CD4+ T cell subsets, macrophages and hematopoietic progenitor cells were analyzed for HIV-1 nucleic acids by droplet digital PCR. RESULTS: Plasma viral loads were reduced by two log10 or to undetectable levels in the 2 and 4ART regimens, respectively. Numbers and distributions of CD4+ memory and regulatory T cells, macrophages and hematopoietic progenitor cells were significantly altered by HIV-1 infection and by both ART regimens. ART reduced viral DNA and RNA in all cell and tissue compartments. While memory cells were the dominant T cell reservoir, integrated HIV-1 DNA was also detected in the BM and spleen macrophages in both regimen-treated mice. CONCLUSION: Despite vigorous ART regimens, HIV-1 DNA and RNA were easily detected in mature macrophages supporting their potential role as an infectious viral reservoir.

Creation of a Long-Acting Nanoformulated 2',3'-Dideoxy-3'-Thiacytidine.

BACKGROUND: Antiretroviral drug discovery and formulation design will facilitate viral clearance in infectious reservoirs. Although progress has been realized for selected hydrophobic integrase and nonnucleoside reverse transcriptase inhibitors, limited success has been seen to date with hydrophilic nucleosides. To overcome these limitations, hydrophobic long-acting drug nanoparticles were created for the commonly used nucleoside reverse transcriptase inhibitor, lamivudine (2',3'-dideoxy-3'-thiacytidine, 3TC). METHODS: A 2-step synthesis created a slow-release long-acting hydrophobic 3TC. Conjugation of 3TC to a fatty acid created a myristoylated prodrug which was encased into a folate-decorated poloxamer 407. Both in vitro antiretroviral efficacy in human monocyte-derived macrophages and pharmacokinetic profiles in mice were evaluated for the decorated nanoformulated drug. RESULTS: A stable drug formulation was produced by poloxamer encasement that improved monocyte-macrophage uptake, antiretroviral activities, and drug pharmacokinetic profiles over native drug formulations. CONCLUSIONS: Sustained release of long-acting antiretroviral therapy is a new therapeutic frontier for HIV/AIDS. 3TC depot formation in monocyte-derived macrophages can be facilitated through stable subcellular internalization and slow drug release.

HIV-1 cellular and tissue replication patterns in infected humanized mice.

Humanized mice have emerged as a testing platform for HIV-1 pathobiology by reflecting natural human disease processes. Their use to study HIV-1 biology, virology, immunology, pathogenesis and therapeutic development has served as a robust alternative to more-well developed animal models for HIV/AIDS. A critical component in reflecting such human pathobiology rests in defining the tissue and cellular sites for HIV-1 infection. To this end, we examined the tissue sites for viral infection in bone marrow, blood, spleens, liver, gut, brain, kidney and lungs of human CD34+ hematopoietic stem cell engrafted virus-infected NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl)/SzJ mice. Cells were analyzed by flow cytometry and sorted from species mixtures defined as CD34+ lineage negative progenitor cells, CD14+CD16+ monocyte-macrophages and central, stem cell and effector memory T cells. The cell distribution and viral life cycle were found dependent on the tissue compartment and time of infection. Cell subsets contained HIV-1 total and integrated DNA as well as multi-spliced and unspliced RNA in divergent proportions. The data support the idea that humanized mice can provide a means to examine the multifaceted sites of HIV-1 replication including, but not limited to progenitor cells and monocyte-macrophages previously possible only in macaques and human.

A new genotype of bovine leukemia virus in South America identified by NGS-based whole genome sequencing and molecular evolutionary genetic analysis.

BACKGROUND: Bovine leukemia virus (BLV) is a member of retroviridae family, together with human T cell leukemia virus types 1 and 2 (HTLV-1 and -2) belonging to the genes deltaretrovirus, and infects cattle worldwide. Previous studies have classified the env sequences of BLV provirus from different geographic locations into eight genetic groups. To investigate the genetic variability of BLV in South America, we performed phylogenetic analyses of whole genome and partial env gp51 sequences of BLV strains isolated from Peru, Paraguay and Bolivia, for which no the molecular characteristics of BLV have previously been published, and discovered a novel BLV genotype, genotype-9, in Bolivia. RESULTS: In Peru and Paraguay, 42.3 % (139/328) and over 50 % (76/139) of samples, respectively, were BLV positive. In Bolivia, the BLV infection rate was up to 30 % (156/507) at the individual level. In Argentina, 325/420 samples were BLV positive, with a BLV prevalence of 77.4 % at the individual level and up to 90.9 % at herd level. By contrast, relatively few BLV positive samples were detected in Chile, with a maximum of 29.1 % BLV infection at the individual level. We performed phylogenetic analyses using two different approaches, maximum likelihood (ML) tree and Bayesian inference, using 35 distinct partial env gp51 sequences from BLV strains isolated from Peru, Paraguay, and Bolivia, and 74 known BLV strains, representing eight different BLV genotypes from various geographical locations worldwide. The results indicated that Peruvian and Paraguayan BLV strains were grouped into genotypes-1, -2, and -6, while those from Bolivia were clustered into genotypes-1, -2, and -6, and a new genotype, genotype-9. Interestingly, these results were confirmed using ML phylogenetic analysis of whole genome sequences obtained by next generation sequencing of 25 BLV strains, assigned to four different genotypes (genotypes-1, -2, -6, and -9) from Peru, Paraguay, and Bolivia. Comparative analyses of complete genome sequences clearly showed some specific substitutions, in both structural and non-structural BLV genes, distinguishing the novel genotype-9 from known genotypes. CONCLUSIONS: Our results demonstrate widespread BLV infection in South American cattle and the existence of a new BLV genotype-9 in Bolivia. We conclude that at least seven BLV genotypes (genotypes-1, -2, -4, -5, -6, -7, and -9) are circulating in South America.

The mixed lineage kinase-3 inhibitor URMC-099 improves therapeutic outcomes for long-acting antiretroviral therapy.

During studies to extend the half-life of crystalline nanoformulated antiretroviral therapy (nanoART) the mixed lineage kinase-3 inhibitor URMC-099, developed as an adjunctive neuroprotective agent was shown to facilitate antiviral responses. Long-acting ritonavir-boosted atazanavir (nanoATV/r) nanoformulations co-administered with URMC-099 reduced viral load and the numbers of HIV-1 infected CD4+ T-cells in lymphoid tissues more than either drug alone in infected humanized NOD/SCID/IL2Rγc-/- mice. The drug effects were associated with sustained ART depots. Proteomics analyses demonstrated that the antiretroviral responses were linked to affected phagolysosomal storage pathways leading to sequestration of nanoATV/r in Rab-associated recycling and late endosomes; sites associated with viral maturation. URMC-099 administered with nanoATV induced a dose-dependent reduction in HIV-1p24 and reverse transcriptase activity. This drug combination offers a unique chemical marriage for cell-based viral clearance. From the Clinical Editor: Although successful in combating HIV-1 infection, the next improvement in antiretroviral therapy (nanoART) would be to devise long acting therapy, such as intra-cellular depots. In this report, the authors described the use of nanoformulated antiretroviral therapy given together with the mixed lineage kinase-3 inhibitor URMC-099, and showed that this combination not only prolonged drug half-life, but also had better efficacy. The findings are hoped to be translated into the clinical setting in the future.