Measuring the latent reservoir for HIV-1: Quantification bias in near full-length genome sequencing methods.

Antiretroviral therapy (ART) effectively inhibits HIV-1 replication but is not curative due to the persistence of a latent viral reservoir in resting CD4+ T cells. This reservoir is a major barrier to cure. Sequencing studies have revealed that the population of proviruses persisting in ART-treated individuals is dominated by defective proviruses that cannot give rise to viral rebound due to fatal defects including large deletions and APOBEC3-mediated hypermutation. Near full genome sequencing (nFGS) of individual proviruses is used in reservoir assays to provide an estimate of the fraction of proviruses that are intact. nFGS methods rely on a long-distance outer PCR capturing most (~9 kb) of the genome, followed by nested inner PCRs. The outer PCR is carried out at limit dilution, and interpretation of the results is based on the assumption that all proviruses are quantitatively captured. Here, we evaluate nFGS methods using the intact proviral DNA assay (IPDA), a multiplex digital droplet PCR assay that quantitates intact and defective proviruses with single molecule sensitivity using only short, highly efficient amplicons. We analyzed proviral templates of known sequence to avoid the additional complication of sequence polymorphism. With the IPDA, we quantitated molecular yields at each step of nFGS methods. We demonstrate that nFGS methods are inefficient and miss ~70% of full-length proviruses due to amplification failure at the initial outer PCR step. In contrast, proviruses with large internal deletions encompassing 70% of the genome can be quantitatively amplified under the same conditions. Accurate measurement of the latent reservoir of HIV-1 is essential for evaluating the efficacy of cure strategies, and the bias against full length proviruses in nFGS methods must be considered.

CD4+ T cells with latent HIV-1 have reduced proliferative responses to T cell receptor stimulation.

The latent reservoir for HIV-1 in resting CD4+ T cells persists despite antiretroviral therapy as a barrier to cure. The antigen-driven proliferation of infected cells is a major mechanism of reservoir persistence. However, activation through the T cell antigen receptor (TCR) can induce latent proviruses, leading to viral cytopathic effects and immune clearance. In single-cell studies, we show that, relative to uninfected cells or cells with a defective provirus, CD4+ T cells with an intact provirus have a profound proliferative defect in response to TCR stimulation. Virion production was observed in only 16.5% of cultures with an intact provirus, but proliferation was reduced even when no virion production was detected. Proliferation was inversely correlated with in vivo clone size. These results may reflect the effects of previous in vivo proliferation and do not support attempts to reduce the reservoir with antiproliferative agents, which may have greater effects on normal T cell responses.

Bilateral atypical femoral fractures in a patient prescribed denosumab - a case report.

Atypical fractures of the diaphyseal femoral shaft have been reported in the literature at an increasing rate over the past few years. They have been observed mostly in patients who have been on prolonged courses of bisphosphonates, with no reported cases of atypical femoral fractures in those treated with other anti-resorptive medications. A 59 year old woman sustained an atypical fracture of her right femur in March 2013. She had a past medical history of rheumatoid arthritis and osteoporosis. She had been on alendronate but it was discontinued after five years in 1999. She received denosumab by subcutaneous injection in December 2012. At follow up, she complained of pain in her left femur and a radiograph revealed atypical appearances. She was admitted in June 2013 for prophylactic nailing of the left femur. To our knowledge, this is the first reported case of bilateral atypical femoral changes in a patient prescribed denosumab. Given that denosumab has been on the market for a short time period, we expect that the number of these cases will increase with time. We emphasise previous guidance that patients who present with new onset hip or thigh pain should be screened for atypical femoral fractures.