Antiviral Activity of MK-4965, a Novel Nonnucleoside Reverse Transcriptase Inhibitor▿

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are the mainstays of therapy for the treatment of human immunodeficiency virus type 1 (HIV-1) infections. However, the effectiveness of NNRTIs can be hampered by the development of resistance mutations which confer cross-resistance to drugs in the same class. Extensive efforts have been made to identify new NNRTIs that can suppress the replication of the prevalent NNRTI-resistant viruses. MK-4965 is a novel NNRTI that possesses both diaryl ether and indazole moieties. The compound displays potency at subnanomolar concentrations against wild-type (WT), K103N, and Y181C reverse transcriptase (RT) in biochemical assays. MK-4965 is also highly potent against the WT virus and two most prevalent NNRTI-resistant viruses (viruses that harbor the K103N or the Y181C mutation), against which it had 95% effective concentrations (EC95s) of <30 nM in the presence of 10% fetal bovine serum. The antiviral EC95 of MK-4965 was reduced approximately four- to sixfold when it was tested in 50% human serum. Moreover, MK-4965 was evaluated with a panel of 15 viruses with NNRTI resistance-associated mutations and showed a superior mutant profile to that of efavirenz but not to that of etravirine. MK-4965 was similarly effective against various HIV-1 subtypes and viruses containing nucleoside reverse transcriptase inhibitor or protease inhibitor resistance-conferring mutations. A two-drug combination study showed that the antiviral activity of MK-4965 was nonantagonistic with each of the 18 FDA-licensed drugs tested vice versa in the present study. Taken together, these in vitro data show that MK-4965 possesses the desired properties for further development as a new NNRTI for the treatment of HIV-1 infection

Respiratory syncytial virus elicits enriched CD8⁺ T lymphocyte responses in lung compared with blood in African green monkeys

<div><p>Respiratory syncytial virus (RSV) is a leading cause of serious lower respiratory tract disease in young children and older adults throughout the world. Prevention of severe RSV disease through active immunization is optimal but no RSV vaccine has been licensed so far. Immune mechanisms of protection against RSV infection in humans have not been fully established, thus a comprehensive characterization of virus-specific immune responses in a relevant animal model will be beneficial in defining correlates of protection. In this study, we infected juvenile naive AGMs with RSV A2 strain and longitudinally assessed virus-specific humoral and cellular immune responses in both peripheral blood and the respiratory tract. RSV viral loads at nasopharyngeal surfaces and in the lung peaked at around day 5 following infection, and then largely resolved by day 10. Low levels of neutralizing antibody titers were detected in serum, with similar kinetics as RSV fusion (F) protein-binding IgG antibodies. RSV infection induced CD8⁺, but very little CD4⁺, T lymphocyte responses in peripheral blood. Virus-specific CD8⁺ T cell frequencies were ~10 fold higher in bronchoaveolar lavage (BAL) compared to peripheral blood and exhibited effector memory (CD95⁺CD28^-) / tissue resident memory (CD69⁺CD103⁺) T (T_RM) cell phenotypes. The kinetics of virus-specific CD8⁺ T cells emerging in peripheral blood and BAL correlated with declining viral titers, suggesting that virus-specific cellular responses contribute to the clearance of RSV infection. RSV-experienced AGMs were protected from subsequent exposure to RSV infection. Additional studies are underway to understand protective correlates in these seropositive monkeys.</p></div

RSV viral replication in AGMs following infection.

<p>Eight AGMs were infected with RSV A2 strain through intranasal and intratracheal inoculation. RSV viral shedding at nasopharyngeal (NP) mucosal surfaces and viral replication in lung (BAL) were determined at 0, 3, 5, 7, and 10 days following infection. (A). RSV viral loads in NP swab elutes were determined by RSV RT-qPCR (Geomean with 95% confidence interval (CI)). (B). RSV viral loads in BAL were determined by RSV plaque forming assay (Geomean with 95% CI). Dashed lines represent limit of detection.</p

Kinetics of RSV-specific CD8⁺ T cell responses in AGMs following infection.

<p>A second group of eight AGMs were infected with RSV A2 strain and CD8⁺ T cell responses were determined by multiparameter flow cytometry with an expanded antibody panel at 0, 7, 9, 14, 21, and 28 days following infection. (A) PBMC or cells isolated from BAL were stimulated with RSV F protein overlapping peptides for evaluation of cytokine secretions. The percentage of IFN-γ-secreting CD8⁺ T cells were used to represent the magnitude of virus-specific CD8⁺ T cell responses. (B). Frequency of IFN-γ-secreting CD8⁺ T cells in responses to RSV proteins N and M (N+M) overlapping peptides stimulation in PBMC and BAL. (C) Expression of proliferation marker Ki67 in CD8⁺ T lymphocytes from PBMC or BAL. *, p<0.05; **, p<0.01; ***, p<0.001. One-way ANNOVA compared to baseline (day 0) levels.</p

RSV cellular immune responses in AGMs following infection.

<p>RSV-specific cellular immune responses in AGMs at 28 days following infection were determined by multiparameter flow cytometry. Cytokine secretions including IFN-γ, IL-2, and TNF-α of CD4⁺ (A) and CD8⁺ (B) T cells in PBMC specific to various RSV antigen stimulations are depicted (line represents mean). (C). Cytokine secretions of CD8⁺ T cells in lung in response to RSV antigens F and N (line represents mean) were depicted. (D). Comparison of RSV F- and N-specific CD8⁺ T cell responses (represented by IFN-γ-secreting CD8⁺ T cells) in PBMC and in lung (N = 8) with p value for paired t test was depicted. (E). Polyfunctionality of RSV-specific CD8⁺ T cell responses in PBMC and BAL (N = 8) were analyzed by Boolean gating using FlowJo software and graphed using SPICE software. The percentage of RSV N-specific CD8⁺ T cells in PBMC and lung secreting one cytokine, 2 cytokines, or 3 cytokines at the same time are depicted respectively in the pie charts. The absolute magnitude of each population secreting individual cytokines, or combinations of two or three cytokines, are depicted in the scattered dot plots below the pie charts (line represent mean). *, p<0.05, one-way ANNOVA compared to the other populations.</p

La Charente

22 juillet 18831883/07/22 (A12,N5011)-1883/07/22.Appartient à l’ensemble documentaire : PoitouCh