Significance Patients homozygous for the C-C chemokine receptor type 5 (CCR5) gene with 32-bp deletions (Δ32) are resistant to HIV infection. Using the piggyBac technology plus transcription activator-like effector nucleases or clustered regularly interspaced short palindromic repeats-Cas9, the authors report, to our knowledge, for the first time in induced pluripotent stem cells (iPSCs) the efficient and seamless derivation of a homozygous CCR5Δ32 mutation, exactly mimicking the natural mutation. Monocytes and macrophages differentiated from these mutated iPSCs in vitro are resistant to HIV infection. This approach can be applied in the future toward the functional cure of HIV infection. The findings are also of great interest to researchers in many fields who wish to correct or introduce mutations in specific genes.

*Members of the gammaretroviruses-such as murine leukemia viruses (MLVs), most notably XMRV [xenotropic murine leukemia virus (X-MLV)-related virus-have been reported to be present in the blood of patients with chronic fatigue syndrome (CFS). We evaluated blood samples from 61 patients with CFS from a single clinical practice, 43 of whom had previously been identified as XMRV-positive. Our analysis included polymerase chain reaction and reverse transcription polymerase chain reaction procedures for detection of viral nucleic acids and assays for detection of infectious virus and virus-specific antibodies. We found no evidence of XMRV or other MLVs in these blood samples. In addition, we found that these gammaretroviruses were strongly (X-MLV) or partially (XMRV) susceptible to inactivation by sera from CFS patients and healthy controls, which suggested that establishment of a successful MLV infection in humans would be unlikely. Consistent with previous reports, we detected MLV sequences in commercial laboratory reagents. Our results indicate that previous evidence linking XMRV and MLVs to CFS is likely attributable to laboratory contamination. X enotropic retroviruses, first discovered in mice, have the unusual characteristic of being endogenous to animal species, i.e., integrated into the animal's genome, but not able to reinfect cells from that species. However, as the name (xenos, foreign) implies, these viruses can infect cells from other animal species. The xenotropic murine leukemia virus (X-MLV), for example, infects cells from several species including humans but cannot infect many mouse cells (1-3). One particular virus within this group, XMRV (xenotropic murine leukemia virus-related virus), was reported to be present in a subset of human prostate tumors (4) and in blood samples from patients with chronic fatigue syndrome (CFS) (5). Other murine-related gammaretroviruses have also reportedly been detected in CFS patients (6). The infection of humans with these viruses is controversial. Investigators evaluating independent cohorts of CFS patients have failed to detect XMRVor other MLVs (7-12), and contamination of human clinical material (13,14) and reagents (e.g., Taq polymerase) (15) with mouse DNA containing MLV-like sequences has been reported.To investigate these discrepancies in a more direct manner, we performed an extensive virological evaluation of blood samples from two human populations with a clinical diagnosis of CFS (16), many of whom had been diagnosed previously as XMRV-infected. The first (P1) consisted of 41 CFS patients ranging in age from 5 to 73 years who came from a private medical practice (Sierra Internal Medicine, Incline Village, Nevada). Twenty-six of the CFS subjects (63%) were female, and 15 (37%) were male; the female median age was 52 years (range 5 to 72 years), and the male median age was 49 years (range 20 to 73 years). These patients were an unselected, sequentially enrolled population submitted for diagnostic testing to the Wisconsin Viral Research Group (WVRG) and w...

HIV replication is suppressed in vitro by a CD8 + cell noncytotoxic antiviral response (CNAR). This activity directly correlates with an asymptomatic clinical state. The objective of this study was to identify the phenotype of CD8 + cell subsets having strong CNAR activity. CD8 + cell subset frequencies and CNAR levels were measured for human immunodeficiency virus (HIV)-uninfected individuals and three groups of HIV type 1 (HIV-1)-infected individuals: asymptomatic individuals with low-level viremia (vHIV), antiretroviral-drug-treated subjects with undetectable virus levels (TxHIV), and therapy-naïve aviremic elite controllers (EC). CD8 + cells from the vHIV individuals exhibited the highest HIV-suppressing activity and had elevated frequencies of CD45RA − CD27 + and PD-1 + (CD279 + ) cells. Functional assessments of CD8 + cells sorted into distinct subsets established that maximal CNAR activity was mediated by CD45RA − CCR7 − CD27 + and PD-1 + CD8 + cells. T cell receptor (TCR) repertoire profiles of CD8 + cell subsets having strong CNAR activity exhibited increased perturbations in comparison to those of inactive subsets. Together, these studies suggest that CNAR is driven by HIV replication and that this antiviral activity is associated with oligoclonally expanded activated CD8 + cells expressing PD-1 and having a transitional memory cell phenotype. The findings better describe the identity of CD8 + cells showing CNAR and should facilitate the evaluation of this important immune response in studies of HIV pathogenesis, resistance to infection, and vaccine development.

Objective: To assess the in-vitro CCR5---tropic and CXCR4---tropic HIV---1 infectivity of immune cells, particularly macrophages, derived from CCR5 gene---edited induced pluripotent stem cells (iPSCs) obtained from the peripheral blood mononuclear cells (PBMC) of HIV---infected patients on antiretroviral therapy (ART). Design: PBMC were obtained from six patients who had been HIV---infected for over 20 years and were on ART for 1---12 years prior to this study. Methods: The PBMC were derived into iPSCs and genetically edited with TALENs or CRISPR---cas9 endonucleases combined with PiggyBac technology to introduce the naturally occurring 32---bp deletion to the CCR5 gene. These iPSCs were differentiated into macrophages, and subsequently challenged with CCR5---tropic or CCR5/CXCR4 dual--- tropic HIV---1 strains. iPSC derivation, gene editing and immune cell differentiation were done in feeder---free, xeno---free in-vitro conditions. Results: Multiple unedited (wild---type) and CCR5 gene---edited (mutant) iPSCs were derived from patients’ PBMC. When differentiated into immune cells and HIV---1 challenged, mutant iPSC lines were resistant to CCR5---tropic and to some extent to CCR5/CXCR4 dual---tropic HIV---1 infection when compared to wild---type iPSC lines. Conclusion: Our study demonstrates that iPSC---derived, gene---edited immune cells are resistant to distinct HIV---1 strains. These findings have important implications for both in-vitro stem cell development and therapeutic approaches to cure HIV infection.