Broadly neutralizing antibody treatment maintained HIV suppression in children with favorable reservoir characteristics in Botswana.

Broadly neutralizing antibodies (bNAbs) may provide an alternative to standard antiretroviral treatment (ART) for controlling HIV-1 replication and may have immunotherapeutic effects against HIV-1 reservoirs. We conducted a prospective clinical trial with two HIV-1 bNAbs (VRC01LS and 10-1074) in children (n = 25) who had previously initiated small-molecule ART treatment before 7 days of age and who continued treatment for at least 96 weeks. Both bNAbs were dosed intravenously every 4 weeks, overlapping with ART for at least 8 weeks and then continued for up to 24 weeks or until detectable viremia of HIV-1 RNA rose above 400 copies per milliliter in the absence of ART. Eleven (44%) children maintained HIV-1 RNA below 400 copies per milliliter through 24 weeks of bNAb-only treatment; 14 (56%) had detectable viremia above 400 copies per milliliter at a median of 4 weeks. Archived HIV-1 provirus susceptible to 10-1074, lower birth HIV-1 DNA reservoir in peripheral blood mononuclear cells, sustained viral suppression throughout early life, and combined negative qualitative HIV-1 DNA polymerase chain reaction and negative HIV-1 serology at entry were associated with maintaining suppression on bNAbs alone. This proof-of-concept study suggests that bNAbs may represent a promising treatment modality for infants and children living with HIV-1. Future studies using newer bNAb combinations with greater breadth and potency are warranted.

Autofluorescent Artemisinin-Benzimidazole Hybrids via Organo-Click Reaction: Study of Antiviral Properties and Mode of Action in Living Cells.

Drug modification by a fluorescent label is a common tool for studying its mechanism of action with fluorescence microscopy techniques. However, the attachment of a fluorescent label can significantly alter the polarity, solubility, and biological activity of the investigated drug, and, as a result, the studied mechanism of action can be misrepresented. Therefore, developing efficient drugs, which are inherently fluorescent and can be tracked directly in the cell is highly favorable. Here an easy formation of fluorescent hybrid drugs is presented, generated by a combination of two readily available non-fluorescent pharmacophores via a non-cleavable linker using a Ramachary-Bressy-Wang organocatalyzed azide-carbonyl [3+2] cycloaddition (organo-click) reaction. All newly prepared fluorescent compounds showed strong anti-HCMV activity (EC50 down to 0.07±0.00 µM), thus presenting a very promising drug developmental basis compared to the approved drug ganciclovir (EC50 2.60±0.50 µM). Remarkably, in vitro fluorescent imaging investigation of new compounds revealed induced changes in mitochondrial structures, which is a phenotypical hallmark of antiviral activity. This approach opens up new vistas for the easy formation of potent fluorescent drugs from readily available non-fluorescent parent compounds and might facilitate insight into their mode of action in living cells, avoiding the requirement of linkage to external fluorescent markers.

The Artemisinin-Derived Autofluorescent Compound BG95 Exerts Strong Anticytomegaloviral Activity Based on a Mitochondrial Targeting Mechanism.

Human cytomegalovirus (HCMV) is a major human pathogen associated with severe pathology. Current options of antiviral therapy only partly satisfy the needs of a well-tolerated long-term treatment/prophylaxis free from drug-induced viral resistance. Recently, we reported the strong antiviral properties in vitro and in vivo of the broad-spectrum anti-infective drug artesunate and its optimized derivatives. NF-κB signaling was described as a targeting mechanism and additional target proteins have recently been identified. Here, we analyzed the autofluorescent hybrid compound BG95, which could be utilized for intracellular visualization by confocal imaging and a tracking analysis in virus-infected primary human fibroblasts. As an important finding, BG95 accumulated in mitochondria visualized by anti-prohibitin and MitoTracker staining, and induced statistically significant changes of mitochondrial morphology, distinct from those induced by HCMV infection. Notably, mitochondrial membrane potential was found substantially reduced by BG95, an effect apparently counteracting efficient HCMV replication, which requires active mitochondria and upregulated energy levels. This finding was consistent with binding properties of artesunate-like compounds to mitochondrial proteins and thereby suggested a new mechanistic aspect. Combined, the present study underlines an important role of mitochondria in the multifaceted, host-directed antiviral mechanism of this drug class, postulating a new mitochondria-specific mode of protein targeting.

Target verification of artesunate-related antiviral drugs: Assessing the role of mitochondrial and regulatory proteins by click chemistry and fluorescence labeling.

Human cytomegalovirus (HCMV) infection is associated with serious pathology such as transplant rejection or embryonic developmental defects. Antiviral treatment with currently available drugs targeting viral enzymes is often accompanied with severe side effects and the occurrence of drug-resistant viruses. For this reason, novel ways of anti-HCMV therapy focusing on so far unexploited small molecules, targets and mechanisms are intensively studied. Recently, we described the pronounced antiviral activity of the artesunate-related class of trioxane compounds, comprising NF-κB/signaling inhibitors like the trimeric derivative TF27, which proved to be highly active in a nanomolar range both in vitro and in vivo. Here, we extend this analysis by presenting further TF27/artesunate-derived antiviral compounds designed for their specific use in target verification by click chemistry applied in fluorescence labeling and tag affinity strategies. Our main findings are as follows: (i) compounds TF27, BG95, AC98 and AC173 exert strong inhibitory activity against HCMV replication in cultured primary human cells, (ii) autofluorescence activity could be quantitatively detected for BG95 and AC98, and confocal fluorescence imaging revealed accumulation in mitochondria, (iii) postulated cellular targets including mitochondrial proteins were down-regulated upon TF27 treatment, (iv) a click chemistry-based protocol of target enrichment was established, and (v) mass spectrometry-based proteomic analysis, using proteins from HCMV-infected fibroblasts covalently interacting with AC173, revealed a refined list of targets. Combined, data strongly suggest a complex mode of antiviral drug-target interaction of artesunate-related compounds, now highlighting potential roles of mitochondrial, NF-κB pathway proteins, exportins and possibly more. This strategy may further promote antiviral drug development on the basis of pharmacologically optimized trioxane derivatives.