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[This corrects the article DOI: 10.1371/journal.ppat.1009404.].

Broadly neutralizing antibodies (bNAbs) directed to HIV-1 have shown promise at suppressing viremia in animal models. However, the use of bNAbs for the central nervous system (CNS) infection is confounded by poor penetration of the blood brain barrier (BBB). Typically, antibody concentrations in the CNS are extremely low; with levels in cerebrospinal fluid (CSF) only 0.1% of blood concentrations. Using a novel nanotechnology platform, which we term nanocapsules, we show effective transportation of the human bNAb PGT121 across the BBB in infant rhesus macaques upon systemic administration up to 1.6% of plasma concentration. We demonstrate that a single dose of PGT121 encased in nanocapsules when delivered at 48h post-infection delays early acute infection with SHIVSF162P3 in infants, with one of four animals demonstrating viral clearance. Importantly, the nanocapsule delivery of PGT121 improves suppression of SHIV infection in the CNS relative to controls.

Due to the durability and persistence of reservoirs of HIV-1-infected cells, combination antiretroviral therapy (ART) is insufficient in eradicating infection. Achieving HIV-1 cure or sustained remission without ART treatment will require the enhanced and persistent effective antiviral immune responses. Chimeric Antigen Receptor (CAR) T-cells have emerged as a powerful immunotherapy and show promise in treating HIV-1 infection. Persistence, trafficking, and maintenance of function remain to be a challenge in many of these approaches, which are based on peripheral T cell modification. To overcome many of these issues, we have previously demonstrated successful long-term engraftment and production of anti-HIV CAR T cells in modified hematopoietic stem cells (HSCs) in vivo. Here we report the development and in vivo testing of second generation CD4-based CARs (CD4CAR) against HIV-1 infection using a HSCs-based approach. We found that a modified, truncated CD4-based CAR (D1D2CAR) allows better CAR-T cell differentiation from gene modified HSCs, and maintains similar CTL activity as compared to the full length CD4-based CAR. In addition, D1D2CAR does not mediate HIV infection or stimulation mediated by IL-16, suggesting lower risk of off-target effects. Interestingly, stimulatory domains of 4-1BB but not CD28 allowed successful hematopoietic differentiation and improved anti-viral function of CAR T cells from CAR modified HSCs. Addition of 4-1BB to CD4 based CARs led to faster suppression of viremia during early untreated HIV-1 infection. D1D2CAR 4-1BB mice had faster viral suppression in combination with ART and better persistence of CAR T cells during ART. In summary, our data indicate that the D1D2CAR-41BB is a superior CAR, showing better HSC differentiation, viral suppression and persistence, and less deleterious functions compared to the original CD4CAR, and should continue to be pursued as a candidate for clinical study.

In a recent clone-tracking experiment, millions of uniquely tagged hematopoietic stem cells (HSCs) and progenitor cells were autologously transplanted into rhesus macaques and peripheral blood containing thousands of tags were sampled and sequenced over 14 years to quantify the abundance of hundreds to thousands of tags or "clones." Two major puzzles of the data have been observed: consistent differences and massive temporal fluctuations of clone populations. The large sample-to-sample variability can lead clones to occasionally go "extinct" but "resurrect" themselves in subsequent samples. Although heterogeneity in HSC differentiation rates, potentially due to tagging, and random sampling of the animals' blood and cellular demographic stochasticity might be invoked to explain these features, we show that random sampling cannot explain the magnitude of the temporal fluctuations. Moreover, we show through simpler neutral mechanistic and statistical models of hematopoiesis of tagged cells that a broad distribution in clone sizes can arise from stochastic HSC self-renewal instead of tag-induced heterogeneity. The very large clone population fluctuations that often lead to extinctions and resurrections can be naturally explained by a generation-limited proliferation constraint on the progenitor cells. This constraint leads to bursty cell population dynamics underlying the large temporal fluctuations. We analyzed experimental clone abundance data using a new statistic that counts clonal disappearances and provided least-squares estimates of two key model parameters in our model, the total HSC differentiation rate and the maximum number of progenitor-cell divisions.

[This corrects the article DOI: 10.1371/journal.ppat.1006753.].

Chimeric Antigen Receptor (CAR) T-cells have emerged as a powerful immunotherapy for various forms of cancer and show promise in treating HIV-1 infection. However, significant limitations are persistence and whether peripheral T cell-based products can respond to malignant or infected cells that may reappear months or years after treatment remains unclear. Hematopoietic Stem/Progenitor Cells (HSPCs) are capable of long-term engraftment and have the potential to overcome these limitations. Here, we report the use of a protective CD4 chimeric antigen receptor (C46CD4CAR) to redirect HSPC-derived T-cells against simian/human immunodeficiency virus (SHIV) infection in pigtail macaques. CAR-containing cells persisted for more than 2 years without any measurable toxicity and were capable of multilineage engraftment. Combination antiretroviral therapy (cART) treatment followed by cART withdrawal resulted in lower viral rebound in CAR animals relative to controls, and demonstrated an immune memory-like response. We found CAR-expressing cells in multiple lymphoid tissues, decreased tissue-associated SHIV RNA levels, and substantially higher CD4/CD8 ratios in the gut as compared to controls. These results show that HSPC-derived CAR T-cells are capable of long-term engraftment and immune surveillance. This study demonstrates for the first time the safety and feasibility of HSPC-based CAR therapy in a large animal preclinical model.

Anti-retroviral drugs suppress HIV-1 plasma viremia to undetectable levels; however, latent HIV-1 persists in reservoirs within HIV-1-infected patients. The silent provirus can be activated through the use of drugs, including protein kinase C activators and histone deacetylase inhibitors. This "shock" approach is then followed by "kill" of the producing cells either through direct HIV-1-induced cell death or natural immune mechanisms. However, these mechanisms are relatively slow and effectiveness is unclear. Here, we develop an approach to specifically target and kill cells that are activated early in the process of virus production. We utilize a novel nanocapsule technology whereby the ricin A chain is encapsulated in an inactive form within a polymer shell. Specificity for release of the ricin A toxin is conferred by peptide crosslinkers that are sensitive to cleavage by HIV-1 protease. By using well-established latent infection models, J-Lat and U1 cells, we demonstrate that only within an HIV-1-producing cell expressing functional HIV-1 protease will the nanocapsule release its ricin A cargo, shutting down viral and cellular protein synthesis, and ultimately leading to rapid death of the producer cell. Thus, we provide proof of principle for a novel technology to kill HIV-1-producing cells without effects on non-target cells.

Small RNAs, including siRNAs, gRNAs and miRNAs, modulate gene expression and serve as potential therapies for human diseases. Delivery to target cells remains the fundamental limitation for use of these RNAs in humans. To address this challenge, we have developed a nanocapsule delivery technology that encapsulates small DNA molecules encoding RNAs into a small (30 nm) polymer nanocapsule. For proof of concept, we transduced DNA expression cassettes for three small RNAs. In one application, the DNA cassette encodes an shRNA transcriptional unit that downregulates CCR5 and protects from HIV-1 infection. The DNA cassette nanocapsules were further engineered for timed release of the DNA cargo for prolonged knockdown of CCR5. Secondly, the nanocapsules provide an efficient means for delivery of gRNAs in the CRISPR/Cas9 system to mutate integrated HIV-1. Finally, delivery of microRNA-125b to mobilized human CD34+ cells enhances survival and expansion of the CD34+ cells in culture.

The use of siRNAs to knock down gene expression can potentially be an approach to treat various diseases. To avoid siRNA toxicity the less transcriptionally active H1 pol III promoter, rather than the U6 promoter, was proposed for siRNA expression. To identify highly efficacious siRNA sequences, extensive screening is required, since current computer programs may not render ideal results. Here, we used CCR5 gene silencing as a model to investigate a rapid and efficient screening approach. We constructed a chimeric luciferase-CCR5 gene for high-throughput screening of siRNA libraries. After screening approximately 900 shRNA clones, 12 siRNA sequences were identified. Sequence analysis demonstrated that most (11 of the 12 sequences) of these siRNAs did not match those identified by available siRNA prediction algorithms. Significant inhibition of CCR5 in a T-lymphocyte cell line and primary T cells by these identified siRNAs was confirmed using the siRNA lentiviral vectors to infect these cells. The inhibition of CCR5 expression significantly protected cells from R5 HIV-1JRCSF infection. These results indicated that the high-throughput screening method allows efficient identification of siRNA sequences to inhibit the target genes at low levels of expression.

Down-regulation of the HIV-1 coreceptor CCR5 holds significant potential for long-term protection against HIV-1 in patients. Using the humanized bone marrow/liver/thymus (hu-BLT) mouse model which allows investigation of human hematopoietic stem/progenitor cell (HSPC) transplant and immune system reconstitution as well as HIV-1 infection, we previously demonstrated stable inhibition of CCR5 expression in systemic lymphoid tissues via transplantation of HSPCs genetically modified by lentiviral vector transduction to express short hairpin RNA (shRNA). However, CCR5 down-regulation will not be effective against existing CXCR4-tropic HIV-1 and emergence of resistant viral strains. As such, combination approaches targeting additional steps in the virus lifecycle are required. We screened a panel of previously published shRNAs targeting highly conserved regions and identified a potent shRNA targeting the R-region of the HIV-1 long terminal repeat (LTR). Here, we report that human CD4(+) T-cells derived from transplanted HSPC engineered to co-express shRNAs targeting CCR5 and HIV-1 LTR are resistant to CCR5- and CXCR4- tropic HIV-1-mediated depletion in vivo. Transduction with the combination vector suppressed CXCR4- and CCR5- tropic viral replication in cell lines and peripheral blood mononuclear cells in vitro. No obvious cytotoxicity or interferon response was observed. Transplantation of combination vector-transduced HSPC into hu-BLT mice resulted in efficient engraftment and subsequent stable gene marking and CCR5 down-regulation in human CD4(+) T-cells within peripheral blood and systemic lymphoid tissues, including gut-associated lymphoid tissue, a major site of robust viral replication, for over twelve weeks. CXCR4- and CCR5- tropic HIV-1 infection was effectively inhibited in hu-BLT mouse spleen-derived human CD4(+) T-cells ex vivo. Furthermore, levels of gene-marked CD4(+) T-cells in peripheral blood increased despite systemic infection with either CXCR4- or CCR5- tropic HIV-1 in vivo. These results demonstrate that transplantation of HSPCs engineered with our combination shRNA vector may be a potential therapy against HIV disease.

Human induced pluripotent stem cells (hiPSCs) provide new possibilities for regenerative therapies. In order for this potential to be achieved, it is critical to efficiently monitor the differentiation of these hiPSCs into specific lineages. Here, we describe a lentiviral reporter vector sensitive to specific microRNAs (miRNA) to show that a single vector bearing multiple miRNA target sequences conjugated to different reporters can be used to monitor hiPSC formation and subsequent differentiation from human fetal fibroblasts (HFFs). The reporter vector encodes EGFP conjugated to the targets of human embryonic stem cell (hESC) specific miRNAs (miR-302a and miR-302d) and mCherry conjugated to the targets of differentiated cells specific miRNAs (miR-142-3p, miR-155, and miR-223). The vector was used to track reprogramming of HFF to iPSC. HFFs co-transduced with this reporter vector and vectors encoding 4 reprogramming factors (OCT4, SOX2, KLF4 and cMYC) were mostly positive for EGFP (67%) at an early stage of hiPSC formation. EGFP expression gradually disappeared and mCherry expression increased indicating less miRNAs specific to differentiated cells and expression of miRNAs specific to hESCs. Upon differentiation of the hiPSC into embryoid bodies, a large fraction of these hiPSCs regained EGFP expression and some of those cells became single positive for EGFP. Further differentiation into neural lineages showed distinct structures demarcated by either EGFP or mCherry expression. These findings demonstrate that a miRNA dependent reporter vector can be a useful tool to monitor living cells during reprogramming of hiPSC and subsequent differentiation to lineage specific cells.

HIV-1 is restricted for infection of primary quiescent T-cells. After viral entry, reverse transcription is initiated but is not completed. Various hypotheses have been proposed for this cellular restriction including insufficient nucleotide pools and cellular factors, but none have been confirmed as the primary mechanism for restriction. A recent study by Chiu et al. implicates APOBEC3G, an anti-retroviral cytidine deaminase, as the cellular restriction factor. Here, we attempted to confirm these findings using the same strategy as reported by Chiu et al. of siRNA targeting knock-down of APOBEC3G expression. In contrast to the published study, our results do not support a role for APOBEC3G in restriction of HIV-1 in quiescent CD4+ T-cells. In our study, we tested the same siRNA as reported by Chiu et al. as well as two additional siRNAs targeting APOBEC3G, one of which showed 2-fold greater knock-down of APOBEC3G mRNA. However, none of the three siRNAs tested had a discernable effect on enhancing infection by HIV-1 in quiescent CD4+ T-cells. Therefore, we conclude that the primary mechanism of HIV-1 restriction in quiescent CD4+ T-cells remains to be elucidated.