Long lasting control of viral rebound with a new drug ABX464 targeting Rev - mediated viral RNA biogenesis.

BACKGROUND: Current therapies have succeeded in controlling AIDS pandemic. However, there is a continuing need for new drugs, in particular those acting through new and as yet unexplored mechanisms of action to achieve HIV infection cure. We took advantage of the unique feature of proviral genome to require both activation and inhibition of splicing of viral transcripts to develop molecules capable of achieving long lasting effect on viral replication in humanized mouse models through inhibition of Rev-mediated viral RNA biogenesis. RESULTS: Current HIV therapies reduce viral load during treatment but titers rebound after treatment is discontinued. We devised a new drug that has a long lasting effect after viral load reduction. We demonstrate here that ABX464 compromises HIV replication of clinical isolates of different subtypes without selecting for drug resistance in PBMCs or macrophages. ABX464 alone, also efficiently compromised viral proliferation in two humanized mouse models infected with HIV that require a combination of 3TC, Raltegravir and Tenofovir (HAART) to achieve viral inhibition in current protocols. Crucially, while viral load increased dramatically just one week after stopping HAART treatment, only slight rebound was observed following treatment cessation with ABX464 and the magnitude of the rebound was maintained below to that of HAART for two months after stopping the treatment. Using a system to visualize single HIV RNA molecules in living cells, we show that ABX464 inhibits viral replication by preventing Rev-mediated export of unspliced HIV-1 transcripts to the cytoplasm and by interacting with the Cap Binding Complex (CBC). Deep sequencing of viral RNA from treated cells established that retained viral RNA is massively spliced but importantly, normal cellular splicing is unaffected by the drug. Consistently ABX464 is non-toxic in humans and therefore represents a promising complement to current HIV therapies. CONCLUSIONS: ABX464 represents a novel class of anti-HIV molecules with unique properties. ABX464 has a long lasting effect in humanized mice and neutralizes the expression of HIV-1 proviral genome of infected immune cells including reservoirs and it is therefore a promising drug toward a functional cure of HIV.

ABX464 (Obefazimod) Upregulates miR-124 to Reduce Proinflammatory Markers in Inflammatory Bowel Diseases.

Advanced therapies have transformed the treatment of inflammatory bowel disease; however, many patients fail to respond, highlighting the need for therapies tailored to the underlying cell and molecular disease drivers. The first-in-class oral molecule ABX464 (obefazimod), which selectively upregulates miR-124, has demonstrated its ability to be a well-tolerated treatment with rapid and sustained efficacy in patients with ulcerative colitis (UC). Here, we provide evidence that ABX464 affects the immune system in vitro , in the murine model of inflammatory bowel disease, and in patients with UC. In vitro , ABX464 treatment upregulated miR-124 and led to decreases in proinflammatory cytokines including interleukin (IL) 17 and IL6, and in the chemokine CCL2. Consistently, miR-124 expression was upregulated in the rectal biopsies and blood samples of patients with UC, and a parallel reduction in Th17 cells and IL17a levels was observed in serum samples. In a mouse model of induced intestinal inflammation with dextran sulfate sodium, ABX464 reversed the increases in multiple proinflammatory cytokines in the colon and the upregulation of IL17a secretion in the mesenteric lymph nodes. By upregulating miR-124, ABX464 acts as "a physiological brake" of inflammation, which may explain the efficacy of ABX464 with a favorable tolerability and safety profile in patients with UC.

Specific and selective induction of miR-124 in immune cells by the quinoline ABX464: a transformative therapy for inflammatory diseases.

Inflammatory diseases are believed to develop as a result of dysregulated inflammatory responses to environmental factors on susceptible genetic backgrounds. Operating at the level of post-transcriptional gene regulation, miRNAs are a class of endogenous, small noncoding RNAs that can promote downregulation of protein expression by translational repression and/or mRNA degradation of target mRNAs involved in inflammation. MiR-124 is a crucial modulator of inflammation and innate immunity that could provide therapeutic restitution of physiological pathways lost in inflammatory diseases. A recently discovered small quinoline, ABX464, was shown to upregulate miR-124 in human immune cells. In vivo, in a proof-of-concept clinical study, ABX464 showed robust and consistent efficacy in ulcerative colitis (UC). In this review, we examine the current therapeutic options proposed for UC and discuss the drug candidate ABX464 in this context.

Both anti-inflammatory and antiviral properties of novel drug candidate ABX464 are mediated by modulation of RNA splicing.

ABX464 is a first-in-class, clinical-stage, small molecule for oral administration that has shown strong anti-inflammatory effects in the DSS-model for inflammatory bowel disease (IBD) and also prevents replication of the HIV virus. ABX464 which binds to cap binding complex (CBC) has demonstrated safety and efficacy in a phase 2a proof-of-concept clinical trial in patients with Ulcerative colitis. Previously, with limited technologies, it was not possible to quantify the effect of ABX464 on viral and cellular RNA biogenesis. Here, using RNA CaptureSeq and deep sequencing, we report that ABX464 enhances the splicing of HIV RNA in infected PBMCs from six healthy individuals and also the expression and splicing of a single long noncoding RNA to generate the anti-inflammatory miR-124 both ex vivo and in HIV patients. While ABX464 has no effect on pre-mRNA splicing of cellular genes, depletion of CBC complex by RNAi leads to accumulation of intron retention transcripts. These results imply that ABX464 did not inhibit the function of CBC in splicing but rather strengthens it under pathological condition like inflammation and HIV infection. The specific dual ability of ABX464 to generate both anti-inflammatory miR-124 and spliced viral RNA may have applicability for the treatment of both inflammatory diseases and HIV infection.

Specific targeting of the F13L protein by ST-246 affects orthopoxvirus production differently.

BACKGROUND: ST-246 is a potent anti-orthopoxviral molecule targeting the F13L protein of vaccinia virus, which is involved in the wrapping of viruses. The discrepancy in sensitivities of several orthopoxviruses to ST-246 has raised questions about potential differences in their replicative cycles and/or the presence of another drug target. METHODS: Density gradients were used to evaluate the differences between the viral cycles of vaccinia, cowpox and camelpox viruses. Also, to investigate if ST-246 inhibits a single target, we compared its activity to that of small interfering RNAs designed to silence the F13L gene (siF13Ls). RESULTS: We showed that the spread of vaccinia virus involved both intracellular and extracellular enveloped viruses, whereas both cowpox and camelpox viruses seemed to propagate via non-enveloped intracellular forms and cell-associated viral particles. Although ST-246 exerted a clear antiviral activity by interfering with the egress of the virus from infected cells, we observed that cowpox and camelpox viruses, in contrast to vaccinia virus, could be directed towards a lytic cycle under ST-246 treatment. We specifically knocked down the F13L transcripts of vaccinia and camelpox viruses by > 85%, reduced virus progeny by 90% and showed that siF13Ls affect camelpox and vaccinia virus propagation differently. Flow cytometry data validated that ST-246 interfered with the activity of the F13L protein, whereas siF13Ls silenced the F13L gene. CONCLUSIONS: Our observations support that vaccinia, cowpox and camelpox viruses exhibit different levels of sensitivity to ST-246 because of dissimilarities between their ways of propagation, and provide a better understanding of the mode of action of ST-246.

RNA sequencing analysis of activated macrophages treated with the anti-HIV ABX464 in intestinal inflammation.

RNA-Seq enables the generation of extensive transcriptome information providing the capability to characterize transcripts (including alternative isoforms and polymorphism), to quantify expression and to identify differential regulation in a single experiment. To reveal the capacity of new anti-HIV ABX464 candidate in modulating the expression of genes, datasets were generated and validated using RNA-seq approach. This comprehensive dataset will be useful to deepen the comprehensive understanding of the progression of human immunodeficiency virus (HIV) associated with mucosal damage in the gastrointestinal (GI) tract and subsequent inflammation, providing an opportunity to generate new therapies, diagnoses, and preventive strategies.

The Anti-Hiv Candidate Abx464 Dampens Intestinal Inflammation by Triggering Il-22 Production in Activated Macrophages.

The progression of human immunodeficiency virus (HIV) is associated with mucosal damage in the gastrointestinal (GI) tract. This damage enables bacterial translocation from the gut and leads to subsequent inflammation. Dextran sulfate sodium (DSS-exposure) is an established animal model for experimental colitis that was recently shown to recapitulate the link between GI-tract damage and pathogenic features of SIV infection. The current study tested the protective properties of ABX464, a first-in-class anti-HIV drug candidate currently in phase II clinical trials. ABX464 treatment strongly attenuated DSS-induced colitis in mice and produced a long-term protection against prolonged DSS-exposure after drug cessation. Consistently, ABX464 reduced the colonic production of the inflammatory cytokines IL-6 and TNFα as well as that of the chemoattractant MCP-1. However, RNA profiling analysis revealed the capacity of ABX464 to induce the expression of IL-22, a cytokine involved in colitis tissue repair, both in DSS-treated mice and in LPS-stimulated bone marrow-derived macrophages. Importantly, anti-IL-22 antibodies significantly reduced the protective effect of ABX464 on colitis in DSS-treated mice. Because reduced IL-22 production in the gut mucosa is an established factor of HIV and DSS-induced immunopathogenesis, our data suggest that the anti-inflammatory properties of ABX464 warrant exploration in both HIV and inflammatory ulcerative colitis (UC) disease.

Vaccinia virus-induced smallpox postvaccinal encephalitis in case of blood-brain barrier damage.

Smallpox vaccination is the only currently effective mean to combat the threat of variola virus used as a bioterrorism agent, although it is responsible for a rare but serious complication, the postvaccinal encephalitis (PVE). Development of safer vaccines therefore is a high priority as the PVE physiopathology is not well understood to date. If vaccinia virus (VACV) is responsible for PVE by central nervous system (CNS) dissemination, trans-migration of the VACV across the blood-brain barrier (BBB) would be supposed to be essential. Given the complexity of the pathogenesis of vaccinia neurovirulence, an in vitro BBB model was used to explore the mechanism of VACV to induce BBB permeability. Two VACV strains were studied, the neurovirulent Western Reserve strain (VACV-WR) and the vaccine reference Lister strain (VACV-List). A mouse model was also developed to study the ability of these two viral strains to propagate in the brain from the blood compartment, their neurovirulence and their neuropathogenesis. In vitro, the loss of permeability resulted from the tight-junctions disruption was induced by virus replication. The ability of VACV to release infectious particles at the abluminal side suggests the capacity of both VACV strains to migrate across the BBB from the blood to the CNS. In vivo, the virus replication in mice CNS was strain-dependent. The VACV-WR laboratory strain proved to be neuroinvasive and neurovirulent, whereas the VACV-List strain is safe in physiological conditions. Mice PVE was observed only with VACV-WR in the co-infection model, when BBB opening was obtained by lipopolysaccharide (LPS) treatment. This study suggests that VACV is able to cross the BBB but encephalitis occurs only in the presence of a co-infection by bacteria. So, a model of co-infection, mimicked by LPS treatment, could have important implication towards the assessment of neurovirulence of new vaccines.

Phenotypic and genetic diversity of the traditional Lister smallpox vaccine.

As an initial step in the development of a second-generation smallpox vaccine derived from the Lister strain, to be prepared for a variola virus threat, diversity of the traditional vaccine was examined by characterizing a series of ten viral clones. In vitro and in vivo phenotypic studies showed that the biological behavior of the clones diverged from each other and in most cases diverged from the vaccinia virus (VACV) Lister parental population. Taken together, these results demonstrate the heterogeneity of the viral population within the smallpox vaccine and highlight the difficulty in choosing one clone which would meet the current requirements for a safe and effective vaccine candidate.

Genomic sequence of a clonal isolate of the vaccinia virus Lister strain employed for smallpox vaccination in France and its comparison to other orthopoxviruses.

Since 1980 there has been global eradication of smallpox due to the success of the vaccination programme using vaccinia virus (VACV). During the eradication period, distinct VACV strains circulated, the Lister strain being the most commonly employed in Europe. Analysis of the safety of smallpox vaccines has suggested that they display significant heterogeneity. To gain a more detailed understanding of the diversity of VACV strains it is important to determine their genomic sequences. Although the sequences of three isolates of the Japanese Lister original strain (VACV-LO) are available, no analysis of the relationship of any Lister sequence compared to other VACV genomes has been reported. Here, we describe the sequence of a representative clonal isolate of the Lister vaccine (VACV-List) used to inoculate the French population. The coding capacity of VACV-List was compared to other VACV strains. The 201 open reading frames (ORFs) were annotated in the VACV-List genome based on protein size, genomic localization and prior characterization of many ORFs. Eleven ORFs were recognized as pseudogenes as they were truncated or fragmented counterparts of larger ORFs in other orthopoxviruses (OPVs). The VACV-List genome also contains several ORFs that have not been annotated in other VACVs but were found in other OPVs. VACV-List and VACV-LO displayed a high level of nucleotide sequence similarity. Compared to the Copenhagen strain of VACV, the VACV-List sequence diverged in three main regions, one of them corresponding to a substitution in VACV-List with coxpox virus GRI-90 strain ORFs, suggestive of prior genetic exchanges. These studies highlight the heterogeneity between VACV strains and provide a basis to better understand differences in safety and efficacy of smallpox vaccines.