Long-acting lenacapavir acts as an effective preexposure prophylaxis in a rectal SHIV challenge macaque model.

Long-acting antiretroviral agents for preexposure prophylaxis (PrEP) represent a promising new alternative to daily oral regimens for HIV prevention. Lenacapavir (LEN) is a first-in-class long-acting capsid inhibitor approved for the treatment of HIV-1 infection. Here, we assessed the efficacy of LEN for PrEP using a single high-dose simian-human immunodeficiency virus (SHIV) rectal challenge macaque model. In vitro, LEN showed potent antiviral activity against SHIV, as it did for HIV-1. In macaques, a single subcutaneous administration of LEN demonstrated dose proportional increases in and durability of drug plasma levels. A high-dose SHIV inoculum for the PrEP efficacy evaluation was identified via virus titration in untreated macaques. LEN-treated macaques were challenged with high-dose SHIV 7 weeks after drug administration, and the majority remained protected from infection, as confirmed by plasma PCR, cell-associated proviral DNA, and serology testing. Complete protection and superiority to the untreated group was observed among animals whose LEN plasma exposure exceeded its model-adjusted clinical efficacy target at the time of challenge. All infected animals had subprotective LEN concentrations and showed no emergent resistance. These data demonstrate effective SHIV prophylaxis in a stringent macaque model at clinically relevant LEN exposures and support the clinical evaluation of LEN for HIV PrEP in humans.

Long-acting capsid inhibitor protects macaques from repeat SHIV challenges.

Because no currently available vaccine can prevent HIV infection, pre-exposure prophylaxis (PrEP) with antiretrovirals (ARVs) is an important tool for combating the HIV pandemic1,2. Long-acting ARVs promise to build on the success of current PrEP strategies, which must be taken daily, by reducing the frequency of administration3. GS-CA1 is a small-molecule HIV capsid inhibitor with picomolar antiviral potency against a broad array of HIV strains, including variants resistant to existing ARVs, and has shown long-acting therapeutic potential in a mouse model of HIV infection4. Here we show that a single subcutaneous administration of GS-CA1 provides long-term protection against repeated rectal simian-human immunodeficiency virus (SHIV) challenges in rhesus macaques. Whereas all control animals became infected after 15 weekly challenges, a single 300 mg kg-1 dose of GS-CA1 provided per-exposure infection risk reduction of 97% for 24 weeks. Pharmacokinetic analysis showed a correlation between GS-CA1 plasma concentration and protection from SHIV challenges. GS-CA1 levels greater than twice the rhesus plasma protein-adjusted 95% effective concentration conferred 100% protection in this model. These proof-of-concept data support the development of capsid inhibitors as a novel long-acting PrEP strategy in humans.

Two-dose emtricitabine/tenofovir alafenamide plus bictegravir prophylaxis protects macaques against SHIV infection.

OBJECTIVES: Current prophylaxis options for people at risk for HIV infection include two US FDA-approved daily pre-exposure prophylaxis (PrEP) regimens and guidelines for a 2-1-1 event-driven course specifically for men who have sex with men. Despite this, PrEP use rates remain suboptimal, and additional PrEP options may help to improve uptake among diverse populations. Here, we evaluated protective efficacy of two-dose PrEP and two-dose postexposure prophylaxis (PEP) schedules with emtricitabine (FTC)/tenofovir alafenamide (TAF) with or without bictegravir (BIC) in an SHIV macaque model. METHODS: Macaques received one oral dose of 200 mg emtricitabine, 25 mg tenofovir alafenamide and 25-100 mg of bictegravir to establish pharmacokinetic profiles of each drug either in the plasma or the peripheral blood mononuclear cells. Protective efficacy of multiple two-dose PrEP and PEP schedules with FTC/TAF with or without bictegravir was then assessed in two repeat low-dose rectal SHIV challenge studies. RESULTS: The data revealed over 95% per-exposure risk reduction with FTC/TAF PrEP initiated 2 h before the exposure, but a loss of significant protection with treatment initiation postexposure. In contrast, FTC/TAF plus BIC offered complete protection as PrEP and greater than 80% per-exposure risk reduction with treatment initiation up to 24 h postexposure. CONCLUSIONS: Together, these results demonstrate that two-dose schedules can protect macaques against SHIV acquisition and highlight the protective advantage of adding the integrase inhibitor bictegravir to the reverse transcriptase inhibitors emtricitabine and tenofovir alafenamide as part of event-driven prophylaxis.

Origin of rebound virus in chronically SIV-infected Rhesus monkeys following treatment discontinuation.

Viral rebound following antiretroviral therapy (ART) discontinuation in HIV-1-infected individuals is believed to originate from a small pool of CD4+ T cells harboring replication-competent provirus. However, the origin and nature of the rebound virus has remained unclear. Recent studies have suggested that rebound virus does not originate directly from individual latent proviruses but rather from recombination events involving multiple proviruses. Here we evaluate the origin of rebound virus in 16 ART-suppressed, chronically SIV-infected rhesus monkeys following ART discontinuation. We sequence viral RNA and viral DNA in these animals prior to ART initiation, during ART suppression, and following viral rebound, and we compare rebound viral RNA after ART discontinuation with near full-length viral DNA from peripheral blood and lymph node mononuclear cells (PBMC and LNMC) during ART suppression. Sequences of initial rebound viruses closely match viral DNA sequences in PBMC and LNMC during ART suppression. Recombinant viruses are rare in the initial rebound virus populations but arise quickly within 2-4 weeks after viral rebound. These data suggest that intact proviral DNA in PBMC and LNMC during ART suppression is likely the direct origin of viral rebound in chronically SIV-infected rhesus monkeys following ART discontinuation.

PD-1 Blockade and TLR7 Activation Lack Therapeutic Benefit in Chronic Simian Immunodeficiency Virus-Infected Macaques on Antiretroviral Therapy.

Antiretroviral therapy (ART) limits human immunodeficiency virus 1 (HIV-1) replication but does not eliminate the long-lived reservoir established shortly after viral acquisition. A successful HIV cure intervention necessitates either elimination or generation of long-term immune control of the persistent viral reservoir. Immune modulating strategies in conjunction with ART hold promise for achieving cure by inducing viral antigen expression and augmenting infected cell killing. Programmed death-1 (PD-1) blockade is a potential means to both activate and eliminate the latent reservoir by restoring exhausted T cell function. We assessed the therapeutic efficacy of PD-1 blockade, Toll-like receptor 7 (TLR7) activation with the agonist vesatolimod, or a combination of the two agents in chronically simian immunodeficiency virus (SIV)-infected macaques suppressed with ART for more than 2 years. Despite achieving extended anti-PD-1 antibody plasma exposure and TLR7-dependent immune activation after multiple administrations, neither individual treatment nor the combination resulted in changes to viral rebound kinetics following ART interruption or reduction in the SIV reservoir size. Our data in the context of other reports demonstrating improved viral control upon PD-1 blockade suggest that its therapeutic utility may be restricted to specific experimental conditions or treatment times during viral pathogenesis.

Publisher Correction: Antibody and TLR7 agonist delay viral rebound in SHIV-infected monkeys.

In Fig. 4b of this Article, the x-axis labels 'PGT121' and 'GS-9620' were inadvertently swapped in both graphs. In Fig. 5a, b, 'TLR7' should have been 'GS-9620'. These figures have been corrected online.

Antibody and TLR7 agonist delay viral rebound in SHIV-infected monkeys.

The latent viral reservoir is the critical barrier for the development of a cure for HIV-1 infection. Previous studies have shown direct antiviral activity of potent HIV-1 Env-specific broadly neutralizing antibodies (bNAbs) administered when antiretroviral therapy (ART) was discontinued, but it remains unclear whether bNAbs can target the viral reservoir during ART. Here we show that administration of the V3 glycan-dependent bNAb PGT121 together with the Toll-like receptor 7 (TLR7) agonist vesatolimod (GS-9620) during ART delayed viral rebound following discontinuation of ART in simian-human immunodeficiency virus (SHIV)-SF162P3-infected rhesus monkeys in which ART was initiated during early acute infection. Moreover, in the subset of monkeys that were treated with both PGT121 and GS-9620 and that did not show viral rebound after discontinuation of ART, adoptive transfer studies and CD8-depletion studies also did not reveal virus. These data demonstrate the potential of bNAb administration together with innate immune stimulation as a possible strategy for targeting the viral reservoir.

Feasibility and biological rationale of repurposing sunitinib and erlotinib for dengue treatment.

There is an urgent need for strategies to combat dengue virus (DENV) infection; a major global threat. We reported that the cellular kinases AAK1 and GAK regulate intracellular trafficking of multiple viruses and that sunitinib and erlotinib, approved anticancer drugs with potent activity against these kinases, protect DENV-infected mice from mortality. Nevertheless, further characterization of the therapeutic potential and underlying mechanism of this approach is required prior to clinical evaluation. Here, we demonstrate that sunitinib/erlotinib combination achieves sustained suppression of systemic infection at approved dose in DENV-infected IFN-α/ß and IFN-γ receptor-deficient mice. Nevertheless, treatment with these blood-brain barrier impermeable drugs delays, yet does not prevent, late-onset paralysis; a common manifestation in this immunodeficient mouse model but not in humans. Sunitinib and erlotinib treatment also demonstrates efficacy in human primary monocyte-derived dendritic cells. Additionally, DENV infection induces expression of AAK1 transcripts, but not GAK, via single-cell transcriptomics, and these kinases are important molecular targets underlying the anti-DENV effect of sunitinib and erlotinib. Lastly, sunitinib/erlotinib combination alters inflammatory cytokine responses in DENV-infected mice. These findings support feasibility of repurposing sunitinib/erlotinib combination as a host-targeted antiviral approach and contribute to understanding its mechanism of antiviral action.

Single-cell transcriptional dynamics of flavivirus infection.

Dengue and Zika viral infections affect millions of people annually and can be complicated by hemorrhage and shock or neurological manifestations, respectively. However, a thorough understanding of the host response to these viruses is lacking, partly because conventional approaches ignore heterogeneity in virus abundance across cells. We present viscRNA-Seq (virus-inclusive single cell RNA-Seq), an approach to probe the host transcriptome together with intracellular viral RNA at the single cell level. We applied viscRNA-Seq to monitor dengue and Zika virus infection in cultured cells and discovered extreme heterogeneity in virus abundance. We exploited this variation to identify host factors that show complex dynamics and a high degree of specificity for either virus, including proteins involved in the endoplasmic reticulum translocon, signal peptide processing, and membrane trafficking. We validated the viscRNA-Seq hits and discovered novel proviral and antiviral factors. viscRNA-Seq is a powerful approach to assess the genome-wide virus-host dynamics at single cell level.

Anticancer kinase inhibitors impair intracellular viral trafficking and exert broad-spectrum antiviral effects.

Global health is threatened by emerging viral infections, which largely lack effective vaccines or therapies. Targeting host pathways that are exploited by multiple viruses could offer broad-spectrum solutions. We previously reported that AAK1 and GAK, kinase regulators of the host adaptor proteins AP1 and AP2, are essential for hepatitis C virus (HCV) infection, but the underlying mechanism and relevance to other viruses or in vivo infections remained unknown. Here, we have discovered that AP1 and AP2 cotraffic with HCV particles in live cells. Moreover, we found that multiple viruses, including dengue and Ebola, exploit AAK1 and GAK during entry and infectious virus production. In cultured cells, treatment with sunitinib and erlotinib, approved anticancer drugs that inhibit AAK1 or GAK activity, or with more selective compounds inhibited intracellular trafficking of HCV and multiple unrelated RNA viruses with a high barrier to resistance. In murine models of dengue and Ebola infection, sunitinib/erlotinib combination protected against morbidity and mortality. We validated sunitinib- and erlotinib-mediated inhibition of AAK1 and GAK activity as an important mechanism of antiviral action. Additionally, we revealed potential roles for additional kinase targets. These findings advance our understanding of virus-host interactions and establish a proof of principle for a repurposed, host-targeted approach to combat emerging viruses.

Hepatitis C Virus Proteins Interact with the Endosomal Sorting Complex Required for Transport (ESCRT) Machinery via Ubiquitination To Facilitate Viral Envelopment.

Enveloped viruses commonly utilize late-domain motifs, sometimes cooperatively with ubiquitin, to hijack the endosomal sorting complex required for transport (ESCRT) machinery for budding at the plasma membrane. However, the mechanisms underlying budding of viruses lacking defined late-domain motifs and budding into intracellular compartments are poorly characterized. Here, we map a network of hepatitis C virus (HCV) protein interactions with the ESCRT machinery using a mammalian-cell-based protein interaction screen and reveal nine novel interactions. We identify HRS (hepatocyte growth factor-regulated tyrosine kinase substrate), an ESCRT-0 complex component, as an important entry point for HCV into the ESCRT pathway and validate its interactions with the HCV nonstructural (NS) proteins NS2 and NS5A in HCV-infected cells. Infectivity assays indicate that HRS is an important factor for efficient HCV assembly. Specifically, by integrating capsid oligomerization assays, biophysical analysis of intracellular viral particles by continuous gradient centrifugations, proteolytic digestion protection, and RNase digestion protection assays, we show that HCV co-opts HRS to mediate a late assembly step, namely, envelopment. In the absence of defined late-domain motifs, K63-linked polyubiquitinated lysine residues in the HCV NS2 protein bind the HRS ubiquitin-interacting motif to facilitate assembly. Finally, ESCRT-III and VPS/VTA1 components are also recruited by HCV proteins to mediate assembly. These data uncover involvement of ESCRT proteins in intracellular budding of a virus lacking defined late-domain motifs and a novel mechanism by which HCV gains entry into the ESCRT network, with potential implications for other viruses. IMPORTANCE: Viruses commonly bud at the plasma membrane by recruiting the host ESCRT machinery via conserved motifs termed late domains. The mechanism by which some viruses, such as HCV, bud intracellularly is, however, poorly characterized. Moreover, whether envelopment of HCV and other viruses lacking defined late domains is ESCRT mediated and, if so, what the entry points into the ESCRT pathway are remain unknown. Here, we report the interaction network of HCV with the ESCRT machinery and a critical role for HRS, an ESCRT-0 complex component, in HCV envelopment. Viral protein ubiquitination was discovered to be a signal for HRS binding and HCV assembly, thereby functionally compensating for the absence of late domains. These findings characterize how a virus lacking defined late domains co-opts ESCRT to bud intracellularly. Since the ESCRT machinery is essential for the life cycle of multiple viruses, better understanding of this virus-host interplay may yield targets for broad-spectrum antiviral therapies.

Selective Inhibitors of Cyclin G Associated Kinase (GAK) as Anti-Hepatitis C Agents.

Cyclin G associated kinase (GAK) emerged as a promising drug target for the treatment of viral infections. However, no potent and selective GAK inhibitors have been reported in the literature to date. This paper describes the discovery of isothiazolo[5,4-b]pyridines as selective GAK inhibitors, with the most potent congeners displaying low nanomolar binding affinity for GAK. Cocrystallization experiments revealed that these compounds behaved as classic type I ATP-competitive kinase inhibitors. In addition, we have demonstrated that these compounds exhibit a potent activity against hepatitis C virus (HCV) by inhibiting two temporally distinct steps in the HCV life cycle (i.e., viral entry and assembly). Hence, these GAK inhibitors represent chemical probes to study GAK function in different disease areas where GAK has been implicated (including viral infection, cancer, and Parkinson's disease).

A role for host activation-induced cytidine deaminase in innate immune defense against KSHV.

Activation-induced cytidine deaminase (AID) is specifically induced in germinal center B cells to carry out somatic hypermutation and class-switch recombination, two processes responsible for antibody diversification. Because of its mutagenic potential, AID expression and activity are tightly regulated to minimize unwanted DNA damage. Surprisingly, AID expression has been observed ectopically during pathogenic infections. However, the function of AID outside of the germinal centers remains largely uncharacterized. In this study, we demonstrate that infection of human primary naïve B cells with Kaposi's sarcoma-associated herpesvirus (KSHV) rapidly induces AID expression in a cell intrinsic manner. We find that infected cells are marked for elimination by Natural Killer cells through upregulation of NKG2D ligands via the DNA damage pathway, a pathway triggered by AID. Moreover, without having a measurable effect on KSHV latency, AID impinges directly on the viral fitness by inhibiting lytic reactivation and reducing infectivity of KSHV virions. Importantly, we uncover two KSHV-encoded microRNAs that directly regulate AID abundance, further reinforcing the role for AID in the antiviral response. Together our findings reveal additional functions for AID in innate immune defense against KSHV with implications for a broader involvement in innate immunity to other pathogens.

Connective tissue growth factor in regulation of RhoA mediated cytoskeletal tension associated osteogenesis of mouse adipose-derived stromal cells.

BACKGROUND: Cytoskeletal tension is an intracellular mechanism through which cells convert a mechanical signal into a biochemical response, including production of cytokines and activation of various signaling pathways. METHODS/PRINCIPAL FINDINGS: Adipose-derived stromal cells (ASCs) were allowed to spread into large cells by seeding them at a low-density (1,250 cells/cm(2)), which was observed to induce osteogenesis. Conversely, ASCs seeded at a high-density (25,000 cells/cm(2)) featured small cells that promoted adipogenesis. RhoA and actin filaments were altered by changes in cell size. Blocking actin polymerization by Cytochalasin D influenced cytoskeletal tension and differentiation of ASCs. To understand the potential regulatory mechanisms leading to actin cytoskeletal tension, cDNA microarray was performed on large and small ASCs. Connective tissue growth factor (CTGF) was identified as a major regulator of osteogenesis associated with RhoA mediated cytoskeletal tension. Subsequently, knock-down of CTGF by siRNA in ASCs inhibited this osteogenesis. CONCLUSIONS/SIGNIFICANCE: We conclude that CTGF is important in the regulation of cytoskeletal tension mediated ASC osteogenic differentiation.

In vitro expansion of adipose-derived adult stromal cells in hypoxia enhances early chondrogenesis.

Cartilage is an avascular tissue, and chondrocytes in vivo experience a severely hypoxic environment. Using a defined in vitro model of early chondrogenesis, we attempted to enrich for cells with an enhanced ability for chondrogenic differentiation by pre-exposure of mouse adipose-derived adult stromal cells (ADASs) to a hypoxic (2% oxygen) environment. ADASs were subsequently expanded in 2% or 21% oxygen environments, resulting in 2 groups of cells, and then early chondrogenic differentiation was induced at 21% oxygen tension using a 3-dimensional micromass culture system. ADAS chondrogenesis was assessed using Alcian Blue staining for proteoglycans and quantification of sulfated glycosaminoglycans. Osteogenesis of the 2 cell groups was also studied. Two percent oxygen tension profoundly increased the proliferation of ADASs. ADASs expanded in 2% oxygen tension exhibited enhanced early chondrogenic differentiation and diminished osteogenesis, suggesting that the reduced oxygen environment may favor chondroprogenitors. Gene expression analysis suggested that matrix metalloproteinase synthesis was inhibited in cells expanded in 2% oxygen. Furthermore, re-oxygenation of the 2% oxygen-expanded ADASs before differentiation did not significantly affect early chondrogenesis. Thus, priming ADASs with 2% oxygen may have selected for chondrogenic progenitors with an enhanced ability to survive and differentiate. This study is relevant for the future application of cell-based therapies involving cartilage tissue regeneration.

Analysis of the material properties of early chondrogenic differentiated adipose-derived stromal cells (ASC) using an in vitro three-dimensional micromass culture system.

Cartilage is an avascular tissue with only a limited potential to heal and chondrocytes in vitro have poor proliferative capacity. Recently, adipose-derived stromal cells (ASC) have demonstrated a great potential for application to tissue engineering due to their ability to differentiate into cartilage, bone, and fat. In this study, we have utilized a high density three-dimensional (3D) micromass model system of early chondrogenesis with ASC. The material properties of these micromasses showed a significant increase in dynamic and static elastic modulus during the early chondrogenic differentiation process. These data suggest that the 3D micromass culture system represents an in vitro model of early chondrogenesis with dynamic cell signaling interactions associated with the mechanical properties of chondrocyte differentiation.