Evaluation of transduction efficiency in macrophage colony-stimulating factor differentiated human macrophages using HIV-1 based lentiviral vectors

<p>Abstract</p> <p>Background</p> <p>Monocyte-derived macrophages contribute to atherosclerotic plaque formation. Therefore, manipulating macrophage function could have significant therapeutic value. The objective of this study was to determine transduction efficiency of two HIV-based lentiviral vector configurations as delivery systems for the transduction of primary human blood monocyte-derived macrophages.</p> <p>Results</p> <p>Human blood monocytes were transduced using two VSV-G pseudotyped HIV-1 based lentiviral vectors containing EGFP expression driven by either native HIV-LTR (VRX494) or EF1α promoters (VRX1090). Lentiviral vectors were added to cultured macrophages at different times and multiplicities of infection (MOI). Transduction efficiency was assessed using fluorescence microscopy and flow cytometry. Macrophages transduced between 2 and 120 hours after culturing showed the highest transduction efficiency at 2-hours transduction time. Subsequently, cells were transduced 2 hours after culturing at various vector concentrations (MOIs of 5, 10, 25 and 50) to determine the amount of lentiviral vector particles required to maximally transduce human monocyte-derived macrophages. On day 7, all transduced cultures showed EGFP-positive cells by microscopy. Flow cytometric analysis showed with all MOIs a peak shift corresponding to the presence of EGFP-positive cells. For VRX494, transduction efficiency was maximal at an MOI of 25 to 50 and ranged between 58 and 67%. For VRX1090, transduction efficiency was maximal at an MOI of 10 and ranged between 80 and 90%. Thus, transductions performed with VRX1090 showed a higher number of EGFP-positive cells than VRX494.</p> <p>Conclusions</p> <p>This report shows that VSV-G pseudotyped HIV-based lentiviral vectors can efficiently transduce human blood monocyte-derived macrophages early during differentiation using low particle numbers that do not interfere with differentiation of monocytes into macrophages.</p

The immunological footprint of CMV in HIV-1 patients stable on long-term ART

BACKGROUND:Most HIV-infected persons are cytomegalovirus (CMV) seropositive and retain latent virus that can be reactivated by immune activation. Their T cell populations express markers reflecting a late stage of differentiation, but the contributions of HIV and CMV to this profile are unclear. We investigated the immunological "footprint" of CMV in HIV patients who had a history of extreme immunodeficiency but were now stable on antiretroviral therapy (ART).RESULTS:Twenty CMV seropositive HIV patients &gt;50years old with nadir CD4 T-cell counts &lt;200 cells/mul were studied after &gt;12years on ART. 16 CMV seropositive and 9 CMV seronegative healthy controls were included. CMV antibody titres were higher in HIV patients than controls (P&lt;0.001-0.003). Levels of soluble B-cell activating factor (sBAFF) were elevated in patients (P=0.002) and correlated with levels of CMV antibodies (P=0.03-0.002), with no clear relationship in controls. CD8 T-cell IFNgamma responses to the IE1 peptide (VLE) remained elevated in HIV patients (P=0.005). The CD57 + CD45RA + CD27 phenotype of CD8 T-cells correlated with age (r=0.60, P=0.006), antibodies against CMV IE1 protein (r=0.44, P=0.06) and CD4 T-cell IFNgamma response to CMV lysate (r=0.45, P=0.05).CONCLUSIONS:Humoral and T-cell responses to CMV remained elevated in HIV patients after &gt;12years on ART. Age and presence of CMV disease influenced CD8 T-cell phenotypes. Elevated levels of sBAFF may be a consequence of HIV disease and contribute to high titres of CMV antibody

Dysregulated TGF-β Production Underlies the Age-Related Vulnerability to Chikungunya Virus

<div><p>Chikungunya virus (CHIKV) is a re-emerging global pathogen with pandemic potential, which causes fever, rash and debilitating arthralgia. Older adults over 65 years are particularly susceptible to severe and chronic CHIKV disease (CHIKVD), accounting for >90% of all CHIKV-related deaths. There are currently no approved vaccines or antiviral treatments available to limit chronic CHIKVD. Here we show that in old mice excessive, dysregulated TGFβ production during acute infection leads to a reduced immune response and subsequent chronic disease. Humans suffering from CHIKV infection also exhibited high TGFβ levels and a pronounced age-related defect in neutralizing anti-CHIKV antibody production. In vivo reduction of TGFβ levels minimized acute joint swelling, restored neutralizing antibody production and diminished chronic joint pathology in old mice. This study identifies increased and dysregulated TGFβ secretion as one key mechanism contributing to the age-related loss of protective anti-CHIKV-immunity leading to chronic CHIKVD.</p></div