CD4-independent use of the CCR5 receptor by sequential primary SIVsm isolates-2

<p><b>Copyright information:</b></p><p>Taken from "CD4-independent use of the CCR5 receptor by sequential primary SIVsm isolates"</p><p>http://www.retrovirology.com/content/4/1/50</p><p>Retrovirology 2007;4():50-50.</p><p>Published online 23 Jul 2007</p><p>PMCID:PMC1950888.</p><p></p>he CCR5 part is represented as grey and the CXCR4 part as black. B. Mode of CCR5-use was analyzed by infection of U87.CD4 cells expressing CCR5 or chimeric receptors. Length of bars indicates degree of infection and syncytia induction as observed in a light microscope 5 and 7 days after infection. Degree of infection follows a scale from 0 to 5 where 0 is no syncytia and RT negative; 1 was 90% of the wells. RT production was positive in grades ranging from 2 to 5 and in concordance with syncytia induction

CD4-independent use of the CCR5 receptor by sequential primary SIVsm isolates-0

<p><b>Copyright information:</b></p><p>Taken from "CD4-independent use of the CCR5 receptor by sequential primary SIVsm isolates"</p><p>http://www.retrovirology.com/content/4/1/50</p><p>Retrovirology 2007;4():50-50.</p><p>Published online 23 Jul 2007</p><p>PMCID:PMC1950888.</p><p></p>RT/well. The day after infection cultures were washed extensively and fresh medium was added. Infected NP-2/CCR5 cells were followed for syncytia induction up to seven days after infection. RT was analyzed in supernatants from NP-2 cells at day 1 after wash and before start of cocultivation. Cocultivation of NP-2/CCR5 cells with hPBMC was started seven days after infection and virus production was measured after additional 6 days. CD4-independent-HIGH, virus production and/or syncytia induction could be detected directly in NP-2/CCR5 cells (dark grey). CD4-independent-LOW, productive infection in NP-2/CCR5 cells revealed only after cocultivation of infected NP-2/CCR5 cells with hPBMC (light grey). RT was analyzed with undiluted supernatants and therefore values above 1000 pg RT/ml cannot be separated. Detection limit for RT was 50 pg/ml. Values are means of duplicate infections

CD4-independent use of the CCR5 receptor by sequential primary SIVsm isolates-4

<p><b>Copyright information:</b></p><p>Taken from "CD4-independent use of the CCR5 receptor by sequential primary SIVsm isolates"</p><p>http://www.retrovirology.com/content/4/1/50</p><p>Retrovirology 2007;4():50-50.</p><p>Published online 23 Jul 2007</p><p>PMCID:PMC1950888.</p><p></p>time in 13 macaques. Neutralization sensitivity of three isolates (A, 2-week isolates, B, 3 or 4-month isolates and C, late isolates) from each macaque was tested with 1:20 dilution of serum [30]. Neutralization was also performed with autologous serum which gave similar results (data not shown). Neutralization sensitivity was measured using the GHOST(3) cell plaque reduction assay which has a cut-off for neutralization at 30% (marked with a line), that is results below 30% are negative [67]. The majority of newly infected macaques harbored virus populations with a CD4-independent-HIGH (dark grey bars) and neutralization sensitive phenotype. This phenotype gradually changed to become a CD4-independent-LOW (light grey bars) and neutralization resistant (below 30%) virus population. CD4-dependent isolates (white bars) were seen in both neutralization sensitive and neutralization resistant populations. Values are mean neutralization (+/- SD) of two independent assays performed in triplicates

CD4-independent use of the CCR5 receptor by sequential primary SIVsm isolates-1

<p><b>Copyright information:</b></p><p>Taken from "CD4-independent use of the CCR5 receptor by sequential primary SIVsm isolates"</p><p>http://www.retrovirology.com/content/4/1/50</p><p>Retrovirology 2007;4():50-50.</p><p>Published online 23 Jul 2007</p><p>PMCID:PMC1950888.</p><p></p>ysed by 0.001% Triton X-100 followed by one cycle of freeze-thawing step. Lysates were titrated at five-fold dilution steps on hPBMC. Supernatant culture fluids from hPBMC infections were collected at day 7 and production of RT was analyzed with undiluted supernatants. The RT cut-off detection level was 50 pg/ml and values above 1000 pg/ml could not be separated. Dark grey bars represent mean virus production in NP-2/CD4/CCR5 cells and. light grey bars represent virus production in NP-2/CCR5 cells. White bars represent virus production measured by RT in PBMC infected with cell lysates diluted 1:5 from infected NP-2/CCR5 cells. Positive syncytia induction (SI) are indicated with +. Means of RT production in duplicates of infection are indicated

CD4-independent use of the CCR5 receptor by sequential primary SIVsm isolates-3

<p><b>Copyright information:</b></p><p>Taken from "CD4-independent use of the CCR5 receptor by sequential primary SIVsm isolates"</p><p>http://www.retrovirology.com/content/4/1/50</p><p>Retrovirology 2007;4():50-50.</p><p>Published online 23 Jul 2007</p><p>PMCID:PMC1950888.</p><p></p>ell (in 88% of the cultures) and RT production was measured in MDM 15 days after infection. Values are means from at least two experiments with MDM from different donors

Comparison of viral phenotypes of 21 mother-child pairs.

(a)<p>Isolates from corresponding mother-child pairs are indicated by the same number preceded by a letter: A for mothers and B for children. Time of sampling is indicated in parenthesis as months before (-), after (+) or within 1 week from (0) delivery/birth. </p><p>Pairs were grouped according to the mother's virus phenotype, i.e. first those carrying an R5^narrow, than an R5^broad and last an R5X4 virus.</p><p>Samples were used to infect U87.CD4 cells expressing wild type CCR5 or CXCR4, or one of the chimeric receptors FC-1, FC-2, FC-4b, FC-5, FC-6 or FC-7. Experiments were repeated twice. –, means no chimeric receptor is used.</p

Distribution of the viral phenotype of transmitting and non transmitting mothers.

<p>Distribution of R5 (black) <i>vs.</i> R5X4 (white) viruses within all virus phenotypes (n = 59 viruses; p = n.s., Fisher's Exact Test) and of R5^narrow (dark gray) <i>vs.</i> R5^broad (light gray) within the R5 phenotype (n = 49 viruses; p = n.s., Fisher's Exact Test).</p

Schematic representation of the chemokine receptors CCR5 and CXCR4 and the chimeric CCR5/CXCR4 receptors.

<p>Chimeric receptors FC-1, FC-2, FC-4b, FC-5, FC-6 and FC-7 were obtained by replacing successively larger parts of CCR5 with corresponding regions of CXCR4.</p

Clinical and viral characteristics of HIV-1 infected children^(a).

(a)<p>symbol - means that the event has not occurred. n.a. = not available. Mos means months of age. Age of appearance of the different conditions is always indicated in months.</p>(b)<p>Narrow and broad refer to viruses with R5 phenotype. Viruses able to exclusively use wild type CCR5 receptor are defined narrow, whereas those using chimeric receptors besides the wild type CCR5 are defined broad. In parenthesis is indicated the age in months of the virus phenotype determination.</p><p>Statistical analysis: Influence of virus R5^broad phenotype on disease progression including children of group 1 and 2, or children of group 1, 2 and 3: p = 0.0260 and p = 0.0218 (Pearson's chi Square), respectively.</p>(c)<p>Age of entry into clinical or immunological category. Categories are defined according to the Centers for Disease Controls <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003292#pone.0003292-Control2" target="_blank">[21]</a>: CDC 3 = severe immune suppression; CDC B = moderate clinical manifestations; CDC C = severe clinical manifestations.</p>(d)<p>Age of start of mono or dual antiretroviral therapy, not HAART.</p

Mean inhibitory concentration (IC) 50 values for duplicate assays performed with TriMab and virus as indicated in the NeutNet Phase I (P1) and Phase II (P2) study.

<p>The cells are colour coded: green, poor or no neutralization, IC50>25 µg/ml; yellow, IC50 5–25 µg/ml; orange, IC50 1–5 µg/ml; red, IC50<1 µg/ml; white, no results available. Assays are grouped on the basis of several criteria: (1) the use of plasmids or culture supernatants as a source of HIV-1; (2) fusion based assays or infection based assays, either with pseudotyped virus or replication competent virus; and (3) the use of cell lines or PBMC. Laboratories performing the assays are numbered (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036438#pone-0036438-g001" target="_blank">Figure 1</a> for reference) and colour coded: blue, TZMbl assay or PSV/plasmid assays; green, PBMC assays using extracellular p24 as readout; pink, plaque reduction assay. In the listing of viruses, to the left, the cells of X4 viruses are labelled grey, the cells of R5 viruses are white.</p