Thor A. Wagner

Proliferation of cells with HIV integrated into cancer genes contributes to persistent infection

A not-so-random integration for HIV Even in the face of a cocktail of antiretroviral drugs, HIV manages to hang on. It does so by integrating its own genome into those of host cells, where it persists in a latent state. To better understand this process, Wagner et al. determined the sites where HIV integrated into three HIV-infected patients treated with antiretroviral drugs for more than a decade. They found an over-representation of sites where HIV integrated into genes associated with cancer and cell proliferation. Also, multiple cells in the same individual harbored the same integration sites. This suggests that integration into specific genes may drive cell proliferation and viral persistence. Science, this issue p. 570 The site of HIV integration may drive cell proliferation and influence the size of the viral reservoir. Antiretroviral treatment (ART) of HIV infection suppresses viral replication. Yet if ART is stopped, virus reemerges because of the persistence of infected cells. We evaluated the contribution of infected-cell proliferation and sites of proviral integration to HIV persistence. A total of 534 HIV integration sites (IS) and 63 adjacent HIV env sequences were derived from three study participants over 11.3 to 12.7 years of ART. Each participant had identical viral sequences integrated at the same position in multiple cells, demonstrating infected-cell proliferation. Integrations were overrepresented in genes associated with cancer and favored in 12 genes across multiple participants. Over time on ART, a greater proportion of persisting proviruses were in proliferating cells. HIV integration into specific genes may promote proliferation of HIV-infected cells, slowing viral decay during ART.

Efficient modification of CCR5 in primary human hematopoietic cells using a megaTAL nuclease and AAV donor template

Therapeutic coding sequences can be targeted to the CCR5 locus of primary human T cells with high efficiency by using megaTAL nuclease and an AAV donor template. Delete and replace Newer gene-editing methods hold promise for correcting human disease but so far have been hampered by low efficiencies when used in primary cells. To address this issue, Sather et al. have devised a more effective way to both disrupt and replace the CCR5 locus in human T cells, a procedure that has already been shown to improve HIV clearance. Serotype 6 of an adeno-associated viral vector worked particularly well for delivery of megaTAL nucleases and homologous donor templates to primary human T cells, achieving efficient gene-editing rates and little toxicity. The megaTALs generate homology-directed repair (rather than previous efforts, which induce nonhomologous end-joining repair) and so was used for both deletion and accurate replacement of the CCR5 locus. The authors demonstrate that chimeric antigen receptors and an HIV fusion inhibitor inserted into the CCR5 locus ameliorate HIV infection in mice and show that their approach also works in CD34+ hematopoietic precursor cells. Genetic mutations or engineered nucleases that disrupt the HIV co-receptor CCR5 block HIV infection of CD4+ T cells. These findings have motivated the engineering of CCR5-specific nucleases for application as HIV therapies. The efficacy of this approach relies on efficient biallelic disruption of CCR5, and the ability to efficiently target sequences that confer HIV resistance to the CCR5 locus has the potential to further improve clinical outcomes. We used RNA-based nuclease expression paired with adeno-associated virus (AAV)–mediated delivery of a CCR5-targeting donor template to achieve highly efficient targeted recombination in primary human T cells. This method consistently achieved 8 to 60% rates of homology-directed recombination into the CCR5 locus in T cells, with over 80% of cells modified with an MND-GFP expression cassette exhibiting biallelic modification. MND-GFP–modified T cells maintained a diverse repertoire and engrafted in immune-deficient mice as efficiently as unmodified cells. Using this method, we integrated sequences coding chimeric antigen receptors (CARs) into the CCR5 locus, and the resulting targeted CAR T cells exhibited antitumor or anti-HIV activity. Alternatively, we introduced the C46 HIV fusion inhibitor, generating T cell populations with high rates of biallelic CCR5 disruption paired with potential protection from HIV with CXCR4 co-receptor tropism. Finally, this protocol was applied to adult human mobilized CD34+ cells, resulting in 15 to 20% homologous gene targeting. Our results demonstrate that high-efficiency targeted integration is feasible in primary human hematopoietic cells and highlight the potential of gene editing to engineer T cell products with myriad functional properties.

An Increasing Proportion of Monotypic HIV-1 DNA Sequences During Antiretroviral Treatment Suggests Proliferation of HIV-Infected Cells

ABSTRACT Understanding how HIV-1 persists during effective antiretroviral therapy (ART) should inform strategies to cure HIV-1 infection. We hypothesize that proliferation of HIV-1-infected cells contributes to persistence of HIV-1 infection during suppressive ART. This predicts that identical or monotypic HIV-1 DNA sequences will increase over time during ART. We analyzed 1,656 env and pol sequences generated following single-genome amplification from the blood and sputum of six individuals during long-term suppressive ART. The median proportion of monotypic sequences increased from 25.0% prior to ART to 43.2% after a median of 9.8 years of suppressive ART. The proportion of monotypic sequences was estimated to increase 3.3% per year (95% confidence interval, 2.3 to 4.4%; P < 0.001). Drug resistance mutations were not more common in the monotypic sequences, arguing against viral replication during times with lower antiretroviral concentrations. Bioinformatic analysis found equivalent genetic distances of monotypic and nonmonotypic sequences from the predicted founder virus sequence, suggesting that the relative increase in monotypic variants over time is not due to selective survival of cells with viruses from the time of acute infection or from just prior to ART initiation. Furthermore, while the total HIV-1 DNA load decreased during ART, the calculated concentration of monotypic sequences was stable in children, despite growth over nearly a decade of observation, consistent with proliferation of infected CD4+ T cells and slower decay of monotypic sequences. Our findings suggest that proliferation of cells with proviruses is a likely mechanism of HIV-1 DNA persistence, which should be considered when designing strategies to eradicate HIV-1 infection.

Association between Detection of HIV-1 DNA Resistance Mutations by a Sensitive Assay at Initiation of Antiretroviral Therapy and Virologic Failure

BACKGROUND Antiretroviral therapy (ART) has become more available throughout the developing world during the past 5 years. The World Health Organization recommends nonnucleoside reverse-transcriptase inhibitor-based regimens as initial ART. However, their efficacy may be compromised by resistance mutations selected by single-dose nevirapine (sdNVP) used to prevent mother-to-child transmission of human immunodeficiency virus (HIV)-1. There is no simple and efficient method to detect such mutations at the initiation of ART. METHODS One hundred eighty-one women who were participating in a clinical trial to prevent mother-to-child transmission and who started NVP-ART after they had received sdNVP or a placebo were included in the study. One hundred copies of each patients HIV-1 DNA were tested for NVP-resistance point-mutations (K103N, Y181C, and G190A) with a sensitive oligonucleotide ligation assay that was able to detect mutants even at low concentrations (> or = 5% of the viral population). Virologic failure was defined as confirmed plasma HIV-1 RNA >50 copies/mL after 6 to 18 months of NVP-ART. RESULTS At initiation of NVP-ART, resistance mutations were identified in 38 (26%) of 148 participants given sdNVP (K103N in 19 [13%], Y181C in 8 [5%], G190A in 28 [19%], and > or = 2 mutations in 15 [10%]), at a median 9.3 months after receipt of sdNVP. The risk of virologic failure was 0.62 (95% confidence interval [CI], 0.46-0.77) in women with > or = 1% resistance mutation, compared with a risk of 0.25 (95% CI, 0.17-0.35) in those without detectable resistance mutations (P < .001). Failure was independently associated with resistance, an interval of <6 months between sdNVP and NVP-ART initiation, and a viral load higher than the median at NVP-ART initiation. CONCLUSIONS Access to simple and inexpensive assays to detect low concentrations of NVP-resistant HIV-1 DNA before the initiation of ART could help improve the outcome of first-line ART.

Successful Treatment of Parainfluenza Virus Respiratory Tract Infection With DAS181 in 4 Immunocompromised Children

Background Parainfluenza virus (PIV), a common pediatric pathogen, is associated with significant morbidity in immunocompromised (IC) hosts. DAS181, a novel sialidase fusion protein inhibitor, seems to be effective against PIV in vitro and in vivo; its use in IC children has not been evaluated. Methods Patients were diagnosed with PIV infection using a quantitative reverse transcription-polymerase chain reaction. DAS181 was obtained under emergency investigational new drug applications and was administered via aerosol chamber or nebulizer. Patients were assessed daily for their clinical condition and adverse outcomes. Results Four pediatric hematopoietic cell transplantation (HCT) patients with PIV detected in respiratory specimens were identified and treated with DAS 181. Patients 1 and 2 were diagnosed with PIV lower respiratory tract infection (LRTI) by bronchoalveolar lavage at 9 months and 2 days after allogeneic transplantation, respectively. Patient 3 was on chemotherapy prior to planned autologous HCT at time of PIV diagnosis from a nasal swab. Patient 4 was diagnosed with PIV via nasal wash 2 days after HCT. Patients 1–3 had clinical symptoms and chest imaging consistent with LRTI. Inhaled DAS181 was administered for 5–10 days. All 4 patients tolerated therapy well. Clinical improvement in oxygen requirement and respiratory rate was observed in all patients who required oxygen at therapy initiation. Viral load decreased in all patients within 1 week of therapy and became undetectable by day 3 of therapy in patient 3. Conclusion DAS181 was used to treat 4 severely IC pediatric patients with PIV disease. The drug was well tolerated. Improvement in both viral loads and symptoms after initiation of therapy was observed in all cases. This report supports prospective, randomized studies in IC patients with PIV infection.

Detection of HIV-1 Drug Resistance in Women Following Administration of a Single Dose of Nevirapine: Comparison of Plasma RNA to Cellular DNA by Consensus Sequencing and by Oligonucleotide Ligation Assay

ABSTRACT A single dose of nevirapine (sdNVP) to prevent mother-to-child transmission of HIV-1 increases the risk of failure of subsequent NVP-containing antiretroviral therapy (ART), especially when initiated within 6 months of sdNVP administration, emphasizing the importance of understanding the decay of nevirapine-resistant mutants. Nevirapine-resistant HIV-1 genotypes (with the mutations K103N, Y181C, and/or G190A) from 21 women were evaluated 10 days and 6 weeks after sdNVP administration and at the initiation of ART. Resistance was assayed by consensus sequencing and by a more sensitive assay (oligonucleotide ligation assay [OLA]) using plasma-derived HIV-1 RNA and cell-associated HIV-1 DNA. OLA detected nevirapine resistance in more specimens than consensus sequencing did (63% versus 33%, P < 0.01). When resistance was detected only by OLA (n = 45), the median mutant concentration was 18%, compared to 61% when detected by both sequencing and OLA (n = 51) (P < 0.0001). The proportion of women whose nevirapine resistance was detected by OLA 10 days after sdNVP administration was higher when we tested their HIV-1 RNA (95%) than when we tested their HIV-1 DNA (88%), whereas at 6 weeks after sdNVP therapy, the proportion was greater with DNA (85%) than with RNA (67%) and remained higher with DNA (33%) than with RNA (11%) at the initiation of antiretroviral treatment (median, 45 weeks after sdNVP therapy). Fourteen women started NVP-ART more than 6 months after sdNVP therapy; resistance was detected by OLA in 14% of the women but only in their DNA. HIV-1 resistance to NVP following sdNVP therapy persists longer in cellular DNA than in plasma RNA, as determined by a sensitive assay using sufficient copies of virus, suggesting that DNA may be superior to RNA for detecting resistance at the initiation of ART.

Detection of HIV-1-Drug-Resistance in Women Following Single-Dose-Nevirapine: Comparison of Plasma RNA versus Cellular DNA by Consensus Sequencing and by Oligonucleotide Ligation Assay

ABSTRACT A single dose of nevirapine (sdNVP) to prevent mother-to-child transmission of HIV-1 increases the risk of failure of subsequent NVP-containing antiretroviral therapy (ART), especially when initiated within 6 months of sdNVP administration, emphasizing the importance of understanding the decay of nevirapine-resistant mutants. Nevirapine-resistant HIV-1 genotypes (with the mutations K103N, Y181C, and/or G190A) from 21 women were evaluated 10 days and 6 weeks after sdNVP administration and at the initiation of ART. Resistance was assayed by consensus sequencing and by a more sensitive assay (oligonucleotide ligation assay [OLA]) using plasma-derived HIV-1 RNA and cell-associated HIV-1 DNA. OLA detected nevirapine resistance in more specimens than consensus sequencing did (63% versus 33%, P < 0.01). When resistance was detected only by OLA (n = 45), the median mutant concentration was 18%, compared to 61% when detected by both sequencing and OLA (n = 51) (P < 0.0001). The proportion of women whose nevirapine resistance was detected by OLA 10 days after sdNVP administration was higher when we tested their HIV-1 RNA (95%) than when we tested their HIV-1 DNA (88%), whereas at 6 weeks after sdNVP therapy, the proportion was greater with DNA (85%) than with RNA (67%) and remained higher with DNA (33%) than with RNA (11%) at the initiation of antiretroviral treatment (median, 45 weeks after sdNVP therapy). Fourteen women started NVP-ART more than 6 months after sdNVP therapy; resistance was detected by OLA in 14% of the women but only in their DNA. HIV-1 resistance to NVP following sdNVP therapy persists longer in cellular DNA than in plasma RNA, as determined by a sensitive assay using sufficient copies of virus, suggesting that DNA may be superior to RNA for detecting resistance at the initiation of ART.

Potential limitation of CCR5 antagonists: drug resistance more often linked to CXCR4-utilizing than to CCR5-utilizing HIV-1.

CCR5 antagonists are approved for treatment-experienced individuals, who are at risk of harboring both drug-resistant and CXCR4-utlizing (X4) HIV-1. If CXCR4 usage and drug resistance are linked, CCR5 antagonists may select for CXCR4-utlizing viruses resistant to antiretrovirals. Analysis of 117 individual viruses found that 69% of CXCR4-utlizing viruses versus 48% of R5 viruses had drug resistance mutations (P = 0.025). Linkage of X4 and drug resistance may limit the effectiveness of CCR5 antagonists.

Increased mutations in Env and Pol suggest greater HIV-1 replication in sputum-derived viruses compared with blood-derived viruses.

Objective:Low-level HIV-1 replication may occur during antiretroviral therapy (ART) that suppresses plasma HIV-1 RNA to less than 50 copies/ml (suppressive ART). Antiretroviral drugs appear less effective in macrophages and monocytes compared with lymphocytes, both in vitro and as implied in vivo by greater viral evolution observed during suppressive ART. Our objective was to examine sputum, which is rich in macrophages, for evidence of increased HIV-1 replication compared with that in the blood during suppressive ART. Design:A cross-sectional study during suppressive ART was performed, and HIV-1 DNA sequences derived from induced sputa and peripheral blood mononuclear cells were compared. Methods:Multiple sequences encoding HIV-1 reverse transcriptase, protease, and envelope were generated using single-genome sequencing. Reverse transcriptase and protease sequences were analyzed for genotypic drug resistance. The evolutionary distances of env sequences from the inferred most recent common ancestor of infection were calculated, and CXCR4 usage was predicted. Results:Nine hundred seventy bidirectional sequences from 11 individuals were analyzed. HIV-1 env and pol derived from sputa had greater frequency of drug-resistance mutations (P = 0.05), evolutionary divergence (P = 0.004), and tendency for CXCR4 usage (P = 0.1) compared with viruses derived from peripheral blood mononuclear cells. Conclusion:The greater frequency of HIV-1 drug-resistance mutations and divergence of HIV-1 env in sputa-derived viruses compared with peripheral blood mononuclear cell-derived viruses suggests greater HIV-1 replication in the respiratory tract compared with the blood. Characterization of viral evolution over time and by cell-type could identify cells that provide a sanctuary for low-level viral replication in the respiratory tract during suppressive ART.

Assessment of mitochondrial toxicity by analysis of mitochondrial protein expression in mononuclear cells

Real‐time PCR has quantified decreased mitochondrial DNA levels in association with nucleoside reverse transcriptase inhibitor (NRTI) therapy of HIV‐infected populations. However, real‐time PCR is best suited to distinguish log differences in an analyte. In an effort to monitor individuals in more detail, we developed a flow cytometric assay to gauge mitochondrial function.