Concanamycin A counteracts HIV-1 Nef to enhance immune clearance of infected primary cells by cytotoxic T lymphocytes.

Nef is an HIV-encoded accessory protein that enhances pathogenicity by down-regulating major histocompatibility class I (MHC-I) expression to evade killing by cytotoxic T lymphocytes (CTLs). A potent Nef inhibitor that restores MHC-I is needed to promote immune-mediated clearance of HIV-infected cells. We discovered that the plecomacrolide family of natural products restored MHC-I to the surface of Nef-expressing primary cells with variable potency. Concanamycin A (CMA) counteracted Nef at subnanomolar concentrations that did not interfere with lysosomal acidification or degradation and were nontoxic in primary cell cultures. CMA specifically reversed Nef-mediated down-regulation of MHC-I, but not CD4, and cells treated with CMA showed reduced formation of the Nef:MHC-I:AP-1 complex required for MHC-I down-regulation. CMA restored expression of diverse allotypes of MHC-I in Nef-expressing cells and inhibited Nef alleles from divergent clades of HIV and simian immunodeficiency virus, including from primary patient isolates. Lastly, we found that restoration of MHC-I in HIV-infected cells was accompanied by enhanced CTL-mediated clearance of infected cells comparable to genetic deletion of Nef. Thus, we propose CMA as a lead compound for therapeutic inhibition of Nef to enhance immune-mediated clearance of HIV-infected cells.

Severe Acute Kidney Injury Following Stage 1 Norwood Palliation: Effect on Outcomes and Risk of Severe Acute Kidney Injury at Subsequent Surgical Stages.

OBJECTIVES: To identify associations of severe acute kidney injury early after stage 1 (Norwood) operation with risk of severe acute kidney injury and comorbidities at subsequent palliative stages in patients with hypoplastic left heart syndrome and other single ventricle lesions with left-sided obstruction. DESIGN: Retrospective cohort study. Severe acute kidney injury defined as Kidney Disease Improving Global Outcomes stage 3. SETTING: Single pediatric cardiac center. PATIENTS: Infants less than or equal to 28 days old with single ventricle physiology and left-sided obstruction undergoing stage 1 operation between September 2007 and November 2012 (n = 136). INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The occurrence rate of severe acute kidney injury was 21% (28/136) following stage 1, 12% (12/98) following stage 2 palliation (superior cavo-pulmonary anastomosis), and 10% (7/73) following stage 3 palliation (total cavo-pulmonary anastomosis). Severe acute kidney injury early after stage 1 operation was significantly associated with continuous intravenous loop diuretic infusion, need for extracorporeal membrane oxygenation, and in-hospital death (all p < 0.05). Gestational age at birth was associated with severe acute kidney injury at stage 2 (p = 0.04) and stage 3 (p = 0.01). Severe acute kidney injury at stage 1 was an independent risk factor for severe acute kidney injury at stage 2 (adjusted odds ratio, 4.3; 95% CI, 1.1-16.9; p = 0.04). Development of severe acute kidney injury after stage 1 was associated with longer mechanical ventilation time after stage 3 (p = 0.047). CONCLUSIONS: Severe acute kidney injury after stage 1 palliation was an independent risk factor for developing severe acute kidney injury at stage 2, and was associated with prolonged duration of mechanical ventilation following stage 3. Information on the incidence and associated risk factors for postoperative acute kidney injury in hypoplastic left heart syndrome patients from multiple congenital heart centers is a necessary next step to further understand the long-term burden of severe acute kidney injury after staged palliation.

Vicriviroc resistance decay and relative replicative fitness in HIV-1 clinical isolates under sequential drug selection pressures.

We previously described an HIV-1-infected individual who developed resistance to vicriviroc (VCV), an investigational CCR5 antagonist, during 28 weeks of therapy (Tsibris AM et al., J. Virol. 82:8210-8214, 2008). To investigate the decay of VCV resistance mutations, a standard clonal analysis of full-length env (gp160) was performed on plasma HIV-1 samples obtained at week 28 (the time of VCV discontinuation) and at three subsequent time points (weeks 30, 42, and 48). During 132 days, VCV-resistant HIV-1 was replaced by VCV-sensitive viruses whose V3 loop sequences differed from the dominant pretreatment forms. A deep-sequencing analysis showed that the week 48 VCV-sensitive V3 loop form emerged from a preexisting viral variant. Enfuvirtide was added to the antiretroviral regimen at week 30; by week 48, enfuvirtide treatment selected for either the G36D or N43D HR-1 mutation. Growth competition experiments demonstrated that viruses incorporating the dominant week 28 VCV-resistant env were less fit than week 0 viruses in the absence of VCV but more fit than week 48 viruses. This week 48 fitness deficit persisted when G36D was corrected by either site-directed mutagenesis or week 48 gp41 domain swapping. The correction of N43D, in contrast, restored fitness relative to that of week 28, but not week 0, viruses. Virus entry kinetics correlated with observed fitness differences; the slower entry of enfuvirtide-resistant viruses corrected to wild-type rates in the presence of enfuvirtide. These findings suggest that while VCV and enfuvirtide select for resistance mutations in only one env subunit, gp120 and gp41 coevolve to maximize viral fitness under sequential drug selection pressures.

ADP ribosylation factor 1 activity is required to recruit AP-1 to the major histocompatibility complex class I (MHC-I) cytoplasmic tail and disrupt MHC-I trafficking in HIV-1-infected primary T cells.

HIV-1-infected cells are partially resistant to anti-HIV cytotoxic T lymphocytes (CTLs) due to the effects of the HIV Nef protein on antigen presentation by major histocompatibility complex class I (MHC-I), and evidence has been accumulating that this function of Nef is important in vivo. HIV Nef disrupts the normal expression of MHC-I by stabilizing a protein-protein interaction between the clathrin adaptor protein AP-1 and the MHC-I cytoplasmic tail. There is also evidence that Nef activates a phosphatidylinositol 3 kinase (PI3K)-dependent GTPase, ADP ribosylation factor 6 (ARF-6), to stimulate MHC-I internalization. However, the relative importance of these two pathways is unclear. Here we report that a GTPase required for AP-1 activity (ARF-1) was needed for Nef to disrupt MHC-I surface levels, whereas no significant requirement for ARF-6 was observed in Nef-expressing T cell lines and in HIV-infected primary T cells. An ARF-1 inhibitor blocked the ability of Nef to recruit AP-1 to the MHC-I cytoplasmic tail, and a dominant active ARF-1 mutant stabilized the Nef-MHC-I-AP-1 complex. These data support a model in which Nef and ARF-1 stabilize an interaction between MHC-I and AP-1 to disrupt the presentation of HIV-1 epitopes to CTLs.

Evolution of CCR5 antagonist resistance in an HIV-1 subtype C clinical isolate.

OBJECTIVES: We previously reported vicriviroc (VCV) resistance in an HIV-infected subject and used deep sequencing and clonal analyses to track the evolution of V3 sequence forms over 28 weeks of therapy. Here, we test the contribution of gp120 mutations to CCR5 antagonist resistance and investigate why certain minority V3 variants emerged as the dominant species under drug pressure. METHODS: Nineteen site-directed HIV-1 mutants were generated that contained gp120 VCV resistance mutations. Viral sensitivities to VCV, maraviroc, TAK-779, and HGS004 were determined. RESULTS: Three patterns of susceptibilities were observed as follows: sigmoid inhibition curves with 50% inhibitory concentration similar to pretreatment virus [07J-week 0 (W0)], single mutants with decreased 50% inhibitory concentrations compared with 07J-W0, and mutants that contained ≥5 of 7 VCV resistance mutations with flattened inhibition curves and decreased or negative percent maximal inhibition. Substitutions such as S306P, which sensitized virus to CCR5 antagonists when present as single mutations, were not detected in the baseline virus population but were necessary for maximal resistance when incorporated into V3 backbones that included preexisting VCV resistance mutations. CONCLUSIONS: CCR5 antagonist resistance was reproduced only when a majority of V3 mutations were present. Minority V3 loop variants may serve as a scaffold upon which additional mutations lead to complete VCV resistance.

In vivo emergence of vicriviroc resistance in a human immunodeficiency virus type 1 subtype C-infected subject.

Little is known about the in vivo development of resistance to human immunodeficiency virus type 1 (HIV-1) CCR5 antagonists. We studied 29 subjects with virologic failure from a phase IIb study of the CCR5 antagonist vicriviroc (VCV) and identified one individual with HIV-1 subtype C who developed VCV resistance. Studies with chimeric envelopes demonstrated that changes within the V3 loop were sufficient to confer VCV resistance. Resistant virus showed VCV-enhanced replication, cross-resistance to another CCR5 antagonist, TAK779, and increased sensitivity to aminooxypentane-RANTES and the CCR5 monoclonal antibody HGS004. Pretreatment V3 loop sequences reemerged following VCV discontinuation, implying that VCV resistance has associated fitness costs.