Summary Human natural killer (NK) cells form a circulating population in a state of dynamic homeostasis. We investigated NK cell homeostasis by labelling dividing cells in vivo using deuterium‐enriched glucose in young and elderly healthy subjects and patients with viral infection. Following a 24‐hr intravenous infusion of 6,6‐D2‐glucose, CD3– CD16+ NK cells sorted from peripheral blood mononuclear cells (PBMC) by fluorescence‐activated cell sorter (FACS) were analysed for DNA deuterium content by gas chromatography mass spectrometry to yield minimum estimates for proliferation rate (p). In healthy young adults (n = 5), deuterium enrichment was maximal ∼ 10 days after labelling, consistent with postmitotic maturation preceding circulation. The mean (± standard deviation) proliferation rate was 4·3 ± 2·4%/day (equivalent to a doubling time of 16 days) and the total production rate was 15 ± 7·6 × 106 cells/l/day. Labelled cells disappeared from the circulation at a similar rate [6·9 ± 4·0%/day; half‐life (T½) < 10 days]. Healthy elderly subjects (n = 8) had lower proliferation and production rates (P = 2·5 ± 1·0%/day and 7·3 ± 3·7 × 106 cells/l/day, respectively; P = 0·04). Similar rates were seen in patients chronically infected with human T‐cell lymphotropic virus type I (HTLV‐I) (P = 3·2 ± 1·9%/day). In acute infectious mononucleosis (n = 5), NK cell numbers were increased but kinetics were unaffected (P = 2·8 ± 1·0%/day) a mean of 12 days after symptom onset. Human NK cells have a turnover time in blood of about 2 weeks. Proliferation rates appear to fall with ageing, remain unperturbed by chronic HTLV‐I infection and normalize rapidly following acute Epstein–Barr virus infection.

Human T-lymphotropic virus type 1 (HTLV-1) is a persistent CD4 ؉ T-lymphotropic retrovirus. Most HTLV-1-infected individuals remain asymptomatic, but a proportion develop adult T cell leukemia or inflammatory disease. It is not fully understood how HTLV-1 persists despite a strong immune response or what determines the risk of HTLV-1-associated diseases. Until recently, it has been difficult to quantify lymphocyte kinetics in humans in vivo. Here, we used deuterated glucose labeling to quantify in vivo lymphocyte dynamics in HTLV-1-infected individuals. We then used these results to address four questions. (i) What is the impact of HTLV-1 infection on lymphocyte dynamics? (ii) How does HTLV-1 persist? (iii) What is the extent of HTLV-1 expression in vivo? (iv) What features of lymphocyte kinetics are associated with HTLV-1-associated myelopathy/tropical spastic paraparesis? We found that CD4 ؉ CD45RO ؉ and CD8 ؉ CD45RO ؉ T lymphocyte proliferation was elevated in HTLV-1-infected subjects compared with controls, with an extra 10 12 lymphocytes produced per year in an HTLV-1-infected subject. The in vivo proliferation rate of CD4 ؉ CD45RO ؉ cells also correlated with ex vivo viral expression. Finally, the inflammatory disease HTLV-1-associated myelopathy/tropical spastic paraparesis was associated with significantly increased CD4 ؉ CD45RO ؉ cell proliferation. We suggest that there is persistent viral gene expression in vivo, which is necessary for the maintenance of the proviral load and determines HTLV-1-associated myelopathy/tropical spastic paraparesis risk. Human T-lymphotropic virus type 1 (HTLV-1) is an exogenous retrovirus that persistently infects 20-30 million people worldwide. It is the etiological agent of adult T cell leukemia and a range of inflammatory diseases including HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a chronic disease of the central nervous system. The majority of HTLV-1-infected individuals remain lifelong asymptomatic carriers (ACs) of the virus.HTLV-1 persists despite the large HTLV-1-specific CD8 ϩ cytotoxic T lymphocyte (CTL) response that is observed in most HTLV-1-infected individuals (1, 2). The observation that cellfree virus, viral protein, and viral mRNA are usually undetectable in the blood (3, 4) suggests that HTLV-1 is largely transcriptionally inactive, at least in the periphery. The viral proteins HBZ, Rex, and p30 II (5-7), as well as epigenetic modifications (8), have been shown to regulate HTLV-1 gene expression and could promote this viral latency. However, the observation of HTLV-1-specific CTL responses (1, 9, 10) indicates that there was certainly viral protein expression in the past and that there is probably a degree of ongoing viral expression. Although HTLV-1 reverse transcriptase has an error rate comparable with other retroviruses (11), the HTLV-1 genome is remarkably stable (12), indicating that very few rounds of replication via reverse transcription have occurred. Thus, it appears that HTLV-1 favors a mode of persistence in which...

Respiratory syncytial virus (RSV) causes respiratory infection in annual epidemics, with infants and the elderly at particular risk of developing severe disease and death. However, despite its importance, no vaccine exists. The chimpanzee adenovirus, PanAd3-RSV, and modified vaccinia virus Ankara, MVA-RSV, are replication defective viral vectors encoding the RSV proteins F, N and M2-1 for the induction of humoral and cellular responses. We performed an open-label, doseescalation, phase 1 clinical trial in 42 healthy adults in which four different combinations of prime/boost vaccinations were investigated for safety and immunogenicity, including both intramuscular and intra-nasal administration of the adenoviral vectored vaccine. The vaccines were safe and well tolerated, with the most common reported adverse events being mild injection site reactions. No vaccine-related serious adverse events occurred. RSV neutralising antibody titres rose in response to intramuscular (IM) prime with PanAd3-RSV, and after IM boost for individuals primed by the intra-nasal (IN) route. Circulating anti-F IgG and IgA antibody secreting cells (ASCs) were observed after IM prime and IM boost. RSV-specific T-cell responses were increased after IM PanAd3-RSV prime and were most efficiently boosted by IM MVA-RSV.Correspondence: christopher.green@paediatrics.ox.uk, tel/fax; 0044 +1865 857 420. Competing interests AJP has previously conducted clinical trials of vaccines on behalf of Oxford University funded by GlaxoSmithKline Biologicals SA and ReiThera S.r.l, but does not receive any personal payments from them. AJP is chair of the UK Department of Health's (DH) Joint Committee on Vaccination and Immunisation (JCVI), but the views expressed in this manuscript do not necessarily represent the views of JCVI or DH. AV, RC, and AN are named inventors on patent applications covering RSV antigen expression system (WO 2012/089833). The remaining authors declare they have no competing interests. IFNγ secretion after boost was from both CD4+ and CD8+ T-cells, without detectable Th2 cytokines that have been previously associated with immune pathogenesis following exposure to RSV after formalin inactivated RSV vaccine. In conclusion, PanAd3-RSV and MVA-RSV are safe and immunogenic in healthy adults. These vaccine candidates warrant further clinical evaluation of efficacy to assess their potential to reduce the burden of RSV disease. Data and materials availability Europe PMC Funders Group

Summary The immune response to human cytomegalovirus (HCMV) infection is characterized by the accumulation of HCMV‐specific CD8+ T cells, particularly in the elderly; such expansions may impair immune responses to other pathogens. We investigated mechanisms underlying HCMV‐specific expansions in 12 young and 21 old healthy subjects (although not all analyses were performed on all subjects). Phenotypically, HCMV‐pentamer+ CD8+ T cells were characterized by marked Vβ restriction, advanced differentiation (being predominantly CD27− CD28−), and variable CD45RO/RA expression. Although more common and larger in older subjects, expansions had similar phenotypic characteristics in the young. In one old subject, repeated studies demonstrated stability in size and Vβ distribution of pentamer+ populations over 6 years. We tested whether HCMV‐specific CD8+ T‐cell expansions arose from accelerated proliferation or extended lifespan by in vivo labelling with deuterated glucose and ex vivo Ki‐67 expression. Uptake of deuterated glucose was lower in pentamer+ cells than in pentamer– CD8+ CD45RO+ or CD8+ CD45RA+ cells in three old subjects, consistent with reduced proliferation and extended lifespan. Similarly Ki‐67 labelling showed no evidence for increased proliferation in HCMV‐specific CD8+ expansions in older subjects, although pentamer– CD45RA+ cells from young donors expressed very little Ki‐67. We investigated Bcl‐2 and CD95 as possible anti‐apoptotic mediators, but neither was associated with pentamer‐positivity. To investigate whether expansion represents a compensatory response to impaired functionality, we performed two tests of functionality, peptide‐stimulated proliferation and CD107 expression; both were intact in pentamer+ cells. Our data suggest that HCMV‐specific CD8+ expansions in older subjects accumulate by extended lifespan, rather than accelerated proliferation.