A Pan-Cyclophilin Inhibitor, CRV431, Decreases Fibrosis and Tumor Development in Chronic Liver Disease Models.

Previous studies show that cyclophilins contribute to many pathologic processes, and cyclophilin inhibitors demonstrate therapeutic activities in many experimental models. However, no drug with cyclophilin inhibition as the primary mode of action has advanced completely through clinical development to market. In this study, we present findings on the cyclophilin inhibitor, CRV431, that highlight its potential as a drug candidate for chronic liver diseases. CRV431 was found to potently inhibit all cyclophilin isoforms tested-A, B, D, and G. Inhibitory constant or IC50 values ranged from 1 to 7 nM, which was up to 13 times more potent than the parent compound, cyclosporine A (CsA), from which CRV431 was derived. Other CRV431 advantages over CsA as a nontransplant drug candidate were significantly diminished immunosuppressive activity, less drug transporter inhibition, and reduced cytotoxicity potential. Oral dosing to mice and rats led to good blood exposures and a 5- to 15-fold accumulation of CRV431 in liver compared with blood concentrations across a wide range of CRV431 dosing levels. Most importantly, CRV431 decreased liver fibrosis in a 6-week carbon tetrachloride model and in a mouse model of nonalcoholic steatohepatitis (NASH). Additionally, CRV431 administration during a late, oncogenic stage of the NASH disease model resulted in a 50% reduction in the number and size of liver tumors. These findings are consistent with CRV431 targeting fibrosis and cancer through multiple, cyclophilin-mediated mechanisms and support the development of CRV431 as a safe and effective drug candidate for liver diseases. SIGNIFICANCE STATEMENT: Cyclophilin inhibitors have demonstrated therapeutic activities in many disease models, but no drug candidates have yet advanced completely through development to market. In this study, CRV431 is shown to potently inhibit multiple cyclophilin isoforms, possess several optimized pharmacological properties, and decrease liver fibrosis and tumors in mouse models of chronic liver disease, which highlights its potential to be the first approved drug primarily targeting cyclophilin isomerases.

C5A Protects Macaques from Vaginal Simian-Human Immunodeficiency Virus Challenge.

A safe and effective vaginal microbicide could decrease human immunodeficiency virus (HIV) transmission in women. Here, we evaluated the safety and microbicidal efficacy of a short amphipathic peptide, C5A, in a rhesus macaque model. We found that a vaginal application of C5A protects 89% of the macaques from a simian-human immunodeficiency virus (SHIV-162P3) challenge. We observed no signs of lesions or inflammation in animals vaginally treated with repeated C5A applications. With its noncellular cytotoxic activity and rare mechanism of action, C5A represents an attractive microbicidal candidate.

Alisporivir inhibition of hepatocyte cyclophilins reduces HBV replication and hepatitis B surface antigen production.

BACKGROUND & AIMS: Cyclophilins are host factors required for hepatitis C virus replication. Cyclophilin inhibitors such as alisporivir have shown strong anti-hepatitis C virus activity in vitro and in clinical studies. However, little is known about whether hepatocyte cyclophilins are involved in the hepatitis B virus (HBV) life cycle. We investigated the effects of 2 cyclophilin inhibitors (alisporivir and NIM811) on HBV replication and hepatitis B surface antigen (HBsAg) production in cell lines. METHODS: Liver-derived cell lines producing full-length HBV and HBsAg particles, owing to stable (HepG2215) or transient (HuH-7) transfection, or infected with HBV (HepaRG cells; Invitrogen [Carlsbad, CA]), were incubated with alisporivir or NIM811 alone, or alisporivir in combination with a direct antiviral (telbivudine). The roles of individual cyclophilins in drug response was evaluated by small interfering RNA knockdown of cyclophilin (CYP)A, CYPC, or CYPD in HepG2215 cells, or CYPA knockdown in HuH-7 cells. The kinetics of antiviral activity were assessed based on levels of HBV DNA and HBsAg and Southern blot analysis. RESULTS: In HepG2215, HuH-7, and HepaRG cells, alisporivir reduced intracellular and secreted HBV DNA, in a dose-dependent manner. Knockdown of CYPA, CYPC, or CYPD (reduced by 80%) significantly reduced levels of HBV DNA and secreted HBsAg. Knockdown of CYPA significantly reduced secretion of HBsAg, leading to accumulation of intracellular HBsAg; the addition of alisporivir greatly reduced levels of HBsAg in these cells. The combination of alisporivir and telbivudine had greater antiviral effects than those of telbivudine or alisporivir alone. CONCLUSIONS: Alisporivir inhibition of cyclophilins in hepatocyte cell lines reduces replication of HBV DNA and HBsAg production and secretion. These effects are potentiated in combination with direct antiviral agents that target HBV-DNA polymerase.

Cyclophilin and NS5A inhibitors, but not other anti-hepatitis C virus (HCV) agents, preclude HCV-mediated formation of double-membrane-vesicle viral factories.

Although the mechanisms of action (MoA) of nonstructural protein 3 inhibitors (NS3i) and NS5B inhibitors (NS5Bi) are well understood, the MoA of cyclophilin inhibitors (CypI) and NS5A inhibitors (NS5Ai) are not fully defined. In this study, we examined whether CypI and NS5Ai interfere with hepatitis C virus (HCV) RNA synthesis of replication complexes (RCs) or with an earlier step of HCV RNA replication, the creation of double-membrane vesicles (DMVs) essential for HCV RNA replication. In contrast to NS5Bi, both CypI and NS5Ai do not block HCV RNA synthesis by way of RCs, suggesting that they exert their antiviral activity prior to the establishment of enzymatically active RCs. We found that viral replication is not a precondition for DMV formation, since the NS3-NS5B polyprotein or NS5A suffices to create DMVs. Importantly, only CypI and NS5Ai, but not NS5Bi, mir-122, or phosphatidylinositol-4 kinase IIIα (PI4KIIIα) inhibitors, prevent NS3-NS5B-mediated DMV formation. NS3-NS5B was unable to create DMVs in cyclophilin A (CypA) knockdown (KD) cells. We also found that the isomerase activity of CypA is absolutely required for DMV formation. This not only suggests that NS5A and CypA act in concert to build membranous viral factories but that CypI and NS5Ai mediate their early anti-HCV effects by preventing the formation of organelles, where HCV replication is normally initiated. This is the first investigation to examine the effect of a large panel of anti-HCV agents on DMV formation, and the results reveal that CypI and NS5Ai act at the same membranous web biogenesis step of HCV RNA replication, thus indicating a new therapeutic target of chronic hepatitis C.

The combination of alisporivir plus an NS5A inhibitor provides additive to synergistic anti-hepatitis C virus activity without detectable cross-resistance.

Alisporivir (ALV), a cyclophilin inhibitor, is a host-targeting antiviral (HTA) with multigenotypic anti-hepatitis C virus (HCV) activity and a high barrier to resistance. Recent advances have supported the concept of interferon (IFN)-free regimens to treat chronic hepatitis C. As the most advanced oral HTA, ALV with direct-acting antivirals (DAAs) represents an attractive drug combination for IFN-free therapy. In this study, we investigated whether particular DAAs exhibit additive, synergistic, or antagonistic effects when combined with ALV. Drug combinations of ALV with NS3 protease, NS5B polymerase, and NS5A inhibitors were investigated in HCV replicons from genotypes 1a, 1b, 2a, 3, and 4a (GT1a to -4a). Combinations of ALV with DAAs exerted an additive effect on GT1 and -4. A significant and specific synergistic effect was observed with ALV-NS5A inhibitor combination on GT2 and -3. Furthermore, ALV was fully active against DAA-resistant variants, and ALV-resistant variants were fully susceptible to DAAs. ALV blocks the contact between cyclophilin A and domain II of NS5A, and NS5A inhibitors target domain I of NS5A; our data suggest a molecular basis for the use of these two classes of inhibitors acting on two distinct domains of NS5A. These results provide in vitro evidence that ALV with NS5A inhibitor combination represents an attractive strategy and a potentially effective IFN-free regimen for treatment of patients with chronic hepatitis C. Due to its high barrier and lack of cross-resistance, ALV could be a cornerstone drug partner for DAAs.

Correlation between NS5A dimerization and hepatitis C virus replication.

Hepatitis C virus (HCV) is the main agent of acute and chronic liver diseases leading to cirrhosis and hepatocellular carcinoma. The current standard therapy has limited efficacy and serious side effects. Thus, the development of alternate therapies is of tremendous importance. HCV NS5A (nonstructural 5A protein) is a pleiotropic protein with key roles in HCV replication and cellular signaling pathways. Here we demonstrate that NS5A dimerization occurs through Domain I (amino acids 1-240). This interaction is not mediated by nucleic acids because benzonase, RNase, and DNase treatments do not prevent NS5A-NS5A interactions. Importantly, DTT abrogates NS5A-NS5A interactions but does not affect NS5A-cyclophilin A interactions. Other reducing agents such as tris(2-carboxyethyl)phosphine and 2-mercaptoethanol also abrogate NS5A-NS5A interactions, implying that disulfide bridges may play a role in this interaction. Cyclophilin inhibitors, cyclosporine A, and alisporivir and NS5A inhibitor BMS-790052 do not block NS5A dimerization, suggesting that their antiviral effects do not involve the disruption of NS5A-NS5A interactions. Four cysteines, Cys-39, Cys-57, Cys-59, and Cys-80, are critical for dimerization. Interestingly, the four cysteines have been proposed to form a zinc-binding motif. Supporting this notion, NS5A dimerization is greatly facilitated by Zn(2+) but not by Mg(2+) or Mn(2+). Importantly, the four cysteines are vital not only for viral replication but also critical for NS5A binding to RNA, revealing a correlation between NS5A dimerization, RNA binding, and HCV replication. Altogether our data suggest that NS5A-NS5A dimerization and/or multimerization could represent a novel target for the development of HCV therapies.

HCV NS5A and IRF9 compete for CypA binding.

BACKGROUND & AIMS: Cyclophilin A (CypA) is vital for HCV replication. Cyp inhibitors successfully decrease viral loads in HCV-infected patients. However, their mechanisms of action remain unknown. Since interferon (IFN) can also suppress HCV replication, we asked whether a link between CypA and the IFN response exists. METHODS: We used cellular and recombinant pulldown approaches to investigate the possibility of a specific association of CypA with host ligands. RESULTS: We found for the first time that CypA binds to a major component of the IFN response - the IFN regulatory factor 9 (IRF9). IRF9 is the DNA-binding component of the transcriptional IFN-stimulated gene factor 3 (ISGF3). CypA binds directly to IRF9 via its peptidyl-prolyl isomerase (PPIase) pocket. Cyp inhibitors such as cyclosporine A (CsA) or non-immunosuppressive derivates such as alisporivir and SCY-635, prevent IRF9-CypA complex formation. CypA binds to the C-terminal IRF-association-domain (IAD), but not to the DNA-binding or linker domains of IRF9. Remarkably, CypA associates with the multimeric ISGF3 complex. We also obtained evidence that CypA neutralization enhances IFN-induced transcription. Interestingly, the hepatitis C virus (HCV) non-structural 5A (NS5A) protein, which is known to modulate the IFN response, competes with IRF9 for CypA binding and can prevent the formation of IRF9-CypA complexes. CONCLUSIONS: This study demonstrates for the first time that CypA binds specifically to a component of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway, IRF9. This study also reveals a novel opportunity of HCV to modulate the IFN response via NS5A.

Sublimable C5A delivery provides sustained and prolonged anti-HIV microbicidal activities.

We have identified a short amphipathic helical peptide, called C5A, which exhibits potent microbicidal activities in vitro and which offers protection from vaginal HIV transmission in vivo in a humanized mouse model. However, there are many obstacles to overcome before C5A can be considered a true microbicidal candidate. First, it must be stabilized against enzymatic degradation in a continuously warm and moist environment. Second, it must be delivered in a controlled manner to achieve long-term and coitally independent efficacy. We demonstrate in this in vitro study that the combination of two matrices with different subliming properties ((hexamethylcyclotrisiloxane [HMCS] and cyclododecane [CDD]) containing 10% labile C5A yielded the best results in terms of controlled release and preserved anti-HIV activity of the peptide when pre-exposed to cell-free medium or cell culture at body temperature for up to 2 months.

The cyclophilin inhibitor SCY-635 disrupts hepatitis C virus NS5A-cyclophilin A complexes.

The nonimmunosuppressive cyclophilin (Cyp) inhibitor SCY-635 blocks hepatitis C virus (HCV) replication both in vitro and in vivo and represents a novel potent anti-HCV agent. However, its mechanism of action remains to be fully elucidated. A growing body of evidence suggests that cyclophilin A (CypA) is absolutely necessary for HCV replication and that the HCV nonstructural 5A (NS5A) protein serves as a main viral ligand for CypA. In this study, we examined the effect of SCY-635 on HCV replication. Specifically, we asked whether SCY-635 blocks HCV replication by targeting CypA-NS5A interactions. We also investigated the possibility that HCV can escape SCY-635 selection pressure and whether this resistance influences either CypA-NS5A interactions or the dependence of HCV on CypA. We found not only that SCY-635 efficiently inhibits HCV replication, but it is sufficient alone to clear HCV replicon-containing cells. We found that SCY-635 prevents CypA-NS5A interactions in a dose-dependent manner. SCY-635 prevents the contact between CypA and NS5A derived from genotypes 1 to 3. Together, these data suggest that NS5A-CypA interactions control HCV replication and that SCY-635 blocks viral replication by preventing the formation of these complexes. We also found that NS5A mutant proteins found in SCY-635-resistant HCV replicons behave similarly to wild-type NS5A in terms of both CypA binding and SCY-635-mediated dissociation and inhibition of CypA binding. However, the NS5A mutations found in SCY-635-resistant HCV replicons rescued viral replication in CypA-knockdown cells, suggesting that the NS5A mutations, which arose in vitro under SCY-635 selection, do not alter the binding affinity of CypA for NS5A. These specific mutations in NS5A eliminate the dependence of HCV RNA replication on the expression of host CypA.

Multiple mutations in hepatitis C virus NS5A domain II are required to confer a significant level of resistance to alisporivir.

Alisporivir is the most advanced host-targeting antiviral cyclophilin (Cyp) inhibitor in phase III studies and has demonstrated a great deal of promise in decreasing hepatitis C virus (HCV) viremia in infected patients. In an attempt to further elucidate the mechanism of action of alisporivir, HCV replicons resistant to the drug were selected. Interestingly, mutations constantly arose in domain II of NS5A. To demonstrate that these mutations are responsible for drug resistance, they were reintroduced into the parental HCV genome, and the resulting mutant viruses were tested for replication in the presence of alisporivir or in the absence of the alisporivir target, CypA. We also examined the effect of the mutations on NS5A binding to itself (oligomerization), CypA, RNA, and NS5B. Importantly, the mutations did not affect any of these interactions. Moreover, the mutations did not preserve NS5A-CypA interactions from alisporivir rupture. NS5A mutations alone render HCV only slightly resistant to alisporivir. In sharp contrast, when multiple NS5A mutations are combined, significant resistance was observed. The introduction of multiple mutations in NS5A significantly restored viral replication in CypA knockdown cells. Interestingly, the combination of NS5A mutations renders HCV resistant to all classes of Cyp inhibitors. This study suggests that a combination of multiple mutations in domain II of NS5A rather than a single mutation is required to render HCV significantly and universally resistant to Cyp inhibitors. This in accordance with in vivo data that suggest that alisporivir is associated with a low potential for development of viral resistance.

Preclinical characterization of naturally occurring polyketide cyclophilin inhibitors from the sanglifehrin family.

Cyclophilin inhibitors currently in clinical trials for hepatitis C virus (HCV) are all analogues of cyclosporine (CsA). Sanglifehrins are a group of naturally occurring cyclophilin binding polyketides that are structurally distinct from the cyclosporines and are produced by a microorganism amenable to biosynthetic engineering for lead optimization and large-scale production by fermentation. Preclinical characterization of the potential utility of this class of compounds for the treatment of HCV revealed that the natural sanglifehrins A to D are all more potent than CsA at disrupting formation of the NS5A-CypA, -CypB, and -CypD complexes and at inhibition of CypA, CypB, and CypD isomerase activity. In particular, sanglifehrin B (SfB) was 30- to 50-fold more potent at inhibiting the isomerase activity of all Cyps tested than CsA and was also shown to be a more potent inhibitor of the 1b subgenomic replicon (50% effective concentrations [EC50s] of 0.070 µM and 0.16 µM in Huh 5-2 and Huh 9-13 cells, respectively). Physicochemical and mouse pharmacokinetic analyses revealed low oral bioavailability (F<4%) and low solubility (<25 µM), although the half-lives (t1/2) of SfA and SfB in mouse blood after intravenous (i.v.) dosing were long (t1/2>5 h). These data demonstrate that naturally occurring sanglifehrins are suitable lead compounds for the development of novel analogues that are less immunosuppressive and that have improved metabolism and pharmacokinetic properties.

Hepatitis C virus NS5A anchor peptide disrupts human immunodeficiency virus.

In the absence of an effective vaccine, there is an urgent need for safe and effective antiviral agents to prevent transmission of HIV. Here, we report that an amphipathic alpha-helical peptide derived from the hepatitis C virus NS5A anchor domain (designated C5A in this article) that has been shown to be virocidal for the hepatitis C virus (HCV) also has potent antiviral activity against HIV. C5A exhibits a broad range of antiviral activity against HIV isolates, and it prevents infection of the three in vivo targets of HIV: CD4(+) T lymphocytes, macrophages, and dendritic cells by disrupting the integrity of the viral membrane and capsid core while preserving the integrity of host membranes. C5A can interrupt an ongoing T cell infection, and it can prevent transmigration of HIV through primary genital epithelial cells, infection of mucosal target cells and transfer from dendritic cells to T cells ex vivo, justifying future experiments to determine whether C5A can prevent HIV transmission in vivo.

The isomerase active site of cyclophilin A is critical for hepatitis C virus replication.

Cyclosporine A and nonimmunosuppressive cyclophilin (Cyp) inhibitors such as Debio 025, NIM811, and SCY-635 block hepatitis C virus (HCV) replication in vitro. This effect was recently confirmed in HCV-infected patients where Debio 025 treatment dramatically decreased HCV viral load, suggesting that Cyps inhibitors represent a novel class of anti-HCV agents. However, it remains unclear how these compounds control HCV replication. Recent studies suggest that Cyps are important for HCV replication. However, a profound disagreement currently exists as to the respective roles of Cyp members in HCV replication. In this study, we analyzed the respective contribution of Cyp members to HCV replication by specifically knocking down their expression by both transient and stable small RNA interference. Only the CypA knockdown drastically decreased HCV replication. The re-expression of an exogenous CypA escape protein, which contains escape mutations at the small RNA interference recognition site, restored HCV replication, demonstrating the specificity for the CypA requirement. We then mutated residues that reside in the hydrophobic pocket of CypA where proline-containing peptide substrates and cyclosporine A bind and that are vital for the enzymatic or the hydrophobic pocket binding activity of CypA. Remarkably, these CypA mutants fail to restore HCV replication, suggesting for the first time that HCV exploits either the isomerase or the chaperone activity of CypA to replicate in hepatocytes and that CypA is the principal mediator of the Cyp inhibitor anti-HCV activity. Moreover, we demonstrated that the HCV NS5B polymerase associates with CypA via its enzymatic pocket. The study of the roles of Cyps in HCV replication should lead to the identification of new targets for the development of alternate anti-HCV therapies.

HCV resistance to cyclosporin A does not correlate with a resistance of the NS5A-cyclophilin A interaction to cyclophilin inhibitors.

BACKGROUND & AIMS: The cyclophilin (Cyp) inhibitors - cyclosporine A (CsA), NIM811, Debio 025, and SCY 635 - block HCV replication both in vitro and in vivo, and represent a novel class of potent anti-HCV agents. We and others showed that HCV relies on cyclophilin A (CypA) to replicate. We demonstrated that the hydrophobic pocket of CypA, where Cyp inhibitors bind, and which controls the isomerase activity of CypA, is critical for HCV replication. Recent studies showed that under Cyp inhibitor selection, mutations arose in the HCV nonstructural 5A (NS5A) protein. This led us to postulate that CypA assists HCV by acting on NS5A. METHODS: We tested this hypothesis by developing several interaction assays including GST pull-down assays, ELISA, and mammalian two-hybrid binding assays. RESULTS: We demonstrated that full-length NS5A and CypA form a stable complex. Remarkably, CsA prevents the CypA-NS5A interaction in a dose-dependent manner. Importantly, the CypA-NS5A interaction is conserved among genotypes and is interrupted by CsA. Surprisingly, the NS5A mutant protein, which arose in CsA-resistant HCV variants, behaves similarly to wild-type NS5A in terms of both CypA binding and CsA-mediated release from CypA. This latter finding suggests that HCV resistance to CsA does not correlate with a resistance of the CypA-NS5A interaction to Cyp inhibitors. Moreover, we found that CypA, devoid of its isomerase activity, fails to bind NS5A. CONCLUSIONS: Altogether these data suggest that CypA, via its isomerase pocket, binds directly to NS5A, and most importantly, that disrupting this interaction stops HCV replication.

Syndecan-Fc hybrid molecule as a potent in vitro microbicidal anti-HIV-1 agent.

In the absence of a vaccine, there is an urgent need for the development of safe and effective topical microbicides to prevent the sexual transmission of human immunodeficiency virus type 1 (HIV-1). In this study, we proposed to develop a novel class of microbicides using syndecan as the antiviral agent. Specifically, we generated a soluble syndecan-Fc hybrid molecule by fusing the ectodomain of syndecan-1 to the Fc domain of a human IgG. We then tested the syndecan-Fc hybrid molecule for various in vitro microbicidal anti-HIV-1 properties. Remarkably, the syndecan-Fc hybrid molecule possesses multiple attractive microbicidal properties: (i) it blocks HIV-1 infection of primary targets including T cells, macrophages, and dendritic cells (DC); (ii) it exhibits a broad range of antiviral activity against primary HIV-1 isolates, multidrug resistant HIV-1 isolates, HIV-2, and simian immunodeficiency virus (SIV); (iii) it prevents transmigration of HIV-1 through human primary genital epithelial cells; (iv) it prevents HIV-1 transfer from dendritic cells to CD4(+) T cells; (v) it is potent when added 2 h prior to addition of HIV-1 to target cells; (vi) it is potent at a low pH; (vii) it blocks HIV-1 infectivity when diluted in genital fluids; and (viii) it prevents herpes simplex virus infection. The heparan sulfate chains of the syndecan-Fc hybrid molecule are absolutely required for HIV-1 neutralization. Several lines of evidence suggest that the highly conserved Arg298 in the V3 region of gp120 serves as the locus for the syndecan-Fc hybrid molecule neutralization. In conclusion, this study suggests that the syndecan-Fc hybrid molecule represents the prototype of a new generation of microbicidal agents that may have promise for HIV-1 prevention.

Cyclophilin A-independent recruitment of NS5A and NS5B into hepatitis C virus replication complexes.

The mechanisms by which cyclophilin A (CypA) governs hepatitis C virus (HCV) replication remain unknown. Since CypA binds two essential components of the HCV replication complex (RC)--the polymerase NS5B and the phosphoprotein NS5A--we asked in this study whether CypA regulates their RC association. We found that CypA, via its isomerase pocket, locates in a protease-resistant compartment similar to that where HCV replicates. CypA association with this compartment is not mediated by HCV. Moreover, CypA depletion of RC does not influence NS5A and NS5B RC association, arguing against a model where CypA governs HCV replication by recruiting NS5A or NS5B into RC.

Structural mapping of CD134 residues critical for interaction with feline immunodeficiency virus.

CD134 is a primary binding receptor for feline immunodeficiency virus (FIV), and with CXCR4 facilitates infection of CD4(+) T cells. Human CD134 fails to support FIV infection. To delineate the regions important for defining virus specificity of CD134, we exchanged domains between human and feline CD134. The binding site for FIV surface glycoprotein (SU) is located in domain 1, in a region distinct from the natural ligand (CD134L)-binding site. Mutagenesis showed that Asp60 and Asp62 are required for interaction with FIV, and modeling studies localized these two residues to the outer edge of domain 1. Substitutions S60D and N62D, in conjunction with H45S, R59G and V64K, imparted both FIV SU binding and receptor function to human CD134. Finally, we demonstrated that soluble CD134 facilitates infection of CD134(-) CXCR4(+) target cells in a manner analogous to CD4 augmentation of HIV infection.

Syndecan-3 is a dendritic cell-specific attachment receptor for HIV-1.

Dendritic cells (DCs) efficiently capture HIV-1 and mediate transmission to T cells, but the underlying molecular mechanism is still being debated. The C-type lectin DC-SIGN is important in HIV-1 transmission by DCs. However, various studies strongly suggest that another HIV-1 receptor on DCs is involved in the capture of HIV-1. Here we have identified syndecan-3 as a major HIV-1 attachment receptor on DCs. Syndecan-3 is a DC-specific heparan sulfate (HS) proteoglycan that captures HIV-1 through interaction with the HIV-1 envelope glycoprotein gp120. Syndecan-3 stabilizes the captured virus, enhances DC infection in cis, and promotes transmission to T cells. Removal of the HSs from the cell surface by heparinase III or by silencing syndecan-3 by siRNA partially inhibited HIV-1 transmission by immature DCs, whereas neutralizing both syndecan-3 and DC-SIGN completely abrogated HIV-1 capture and subsequent transmission. Thus, HIV-1 exploits both syndecan-3 and DC-SIGN to mediate HIV-1 transmission, and an effective microbicide should target both syndecan-3 and DC-SIGN on DCs to prevent transmission.

The inhibitor of apoptosis proteins antagonist Debio 1143 promotes the PD-1 blockade-mediated HIV load reduction in blood and tissues of humanized mice.

The immune checkpoint programmed cell death protein 1 (PD-1) plays a major role in T cell exhaustion in cancer and chronic HIV infection. The inhibitor of apoptosis protein antagonist Debio 1143 (D1143) enhances tumor cell death and synergizes with anti-PD-1 agents to promote tumor immunity and displayed HIV latency reversal activity in vitro. We asked in this study whether D1143 would stimulate the potency of an anti-human PD-1 monoclonal antibody (mAb) to reduce HIV loads in humanized mice. Anti-PD-1 mAb treatment decreased PD-1+ CD8+ cell population by 32.3% after interruption of four weeks treatment, and D1143 co-treatment further reduced it from 32.3 to 73%. Anti-PD-1 mAb administration reduced HIV load in blood by 94%, and addition of D1143 further enhanced this reduction from 94 to 97%. D1143 also more profoundly promoted with the anti-PD-1-mediated reduction of HIV loads in all tissues analyzed including spleen (71 to 96.4%), lymph nodes (64.3 to 80%), liver (64.2 to 94.4), lung (64.3 to 80.1%) and thymic organoid (78.2 to 98.2%), achieving a >5 log reduction of HIV loads in CD4+ cells isolated from tissues 2 weeks after drug treatment interruption. Ex vivo anti-CD3/CD28 stimulation increased the ability to activate exhausted CD8+ T cells in infected mice having received in vivo anti-PD-1 treatment by 7.9-fold (5 to 39.6%), and an additional increase by 1.7-fold upon D1143 co-treatment (39.6 to 67.3%). These findings demonstrate for the first time that an inhibitor of apoptosis protein antagonist enhances in a statistically manner the effects of an immune check point inhibitor on antiviral immunity and on HIV load reduction in tissues of humanized mice, suggesting that the combination of two distinct classes of immunomodulatory agents constitutes a promising anti-HIV immunotherapeutic approach.

The cyclophilin inhibitor CRV431 inhibits liver HBV DNA and HBsAg in transgenic mice.

Hepatitis B virus (HBV) infection is a major health burden worldwide with 240 million chronically infected individuals. Nucleos(t)ide analogs and interferons are the current standards of care due to their suppression of HBV replication, but the treatments rarely eradicate HBV from individuals. Similar to current treatments for human immunodeficiency virus type-1 (HIV-1) and hepatitis C virus (HCV) patients, improved HBV therapies will require the combination of multiple drugs which target distinct steps of the HBV life cycle. In this study, we tested the potential of a cyclophilin inhibitor, CRV431, to affect HBV replication in transgenic mice. We found that oral treatment with CRV431 (50 mg/kg/day) for a period of 16 days significantly reduced liver HBV DNA levels and moderately decreased serum HBsAg levels. We observed an additive inhibitory effect on liver HBV DNA levels in mice treated with a combination of low doses of CRV431 (10 mg/kg/day) and the nucleotide prodrug, tenofovir exalidex (TXL), (5 mg/kg/day). No toxicity was observed in CRV431-treated mice. Although it is well known that CRV431 neutralizes the peptidyl-prolyl isomerase activity of cyclophilins, its anti-HBV mechanism(s) of action remains unknown. Nevertheless, this study provides the first demonstration of a beneficial effect of a cyclophilin inhibitor in vivo in an HBV transgenic mouse model. Altogether our data reveal the potential of CRV431 to be part of improved new therapies for HBV patients.