Kaplan Meier Analysis of 506 seroconverters showing a relation of CCR5Δ32 genotype with a CD4⁺ T-cell count <200 µl⁻¹.

<p>A CD4⁺ T-cell count <200 µl-1 was documented for n = 57 of 422 individuals homozygous for the wildtype CCR5 genotype, and for n = 9 out of 84 CCR5Δ32 heterozygotes. Data censoring was due to introduction of antiretroviral therapy before a CD4 cell count of 200 µl⁻¹ was reached. The difference between both groups implied by the Kaplan-Meier diagram is reflected in a statistical trend (p = 0.1 as calculated by the Log-Rank test).</p

CCR5Δ32 genotype distribution in HIV-positive and HIV-negative study subjects

1<p>2×2 or 3×2 χ2 comparisons, depending on the presence or absence of heterozygous and mutant homozygotes in the respective subgroup. Comparisons were conducted separately according to ethnicity</p

Clinical, immunological and viral load characteristics in an HIV-infected patient homozygous for the CCR5Δ32 deletion (patient#12).

<p>Antiretroviral therapy was initiated twice, eight days after diagnosis with efavirenz (EFV), stavudine (d4T) and lopinavir-ritonavir (LPVr) and again, following rapid disease progression, 18 months after diagnosis with Efavirenz and Zidovudine (AZT) plus Lamivudine (3TC). Unbroken line with closed circles: CD4⁺ T-cell counts. Unbroken line with closed diamonds: Plasma viral loads. Broken line with closed circles: CD8⁺ T-cell counts. Blue arrow: negative HIV-1 antibody test. Red arrow: positive HIV-1 antibody test. Black arrows: Proctitis (1) / Shigellosis (2).</p

Relation of CCR5Δ32 genotype with viral setpoints in 319 Caucasian seroconverters.

<p>Scattered dots represent individual viral setpoints (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002747#s4" target="_blank">Material and Methods</a> for a definition), whereas vertical lines demarcate the median in each genetic group.</p

Demographic characteristics of HIV positive and HIV negative studied subjects

1<p>Age median (IR, interquantile range) in years.</p>2<p>Of these, n = 2 individuals were occupationally exposed to HIV; risk group is unknown for n = 14 individuals.</p

The Attenuated <i>Brucella abortus</i> Strain 19 Invades, Persists in, and Activates Human Dendritic Cells, and Induces the Secretion of IL-12p70 but Not IL-23

<div><p>Background</p><p>Bacterial vectors have been proposed as novel vaccine strategies to induce strong cellular immunity. Attenuated strains of <i>Brucella abortus</i> comprise promising vector candidates since they have the potential to induce strong CD4⁺ and CD8⁺ T-cell mediated immune responses in the absence of excessive inflammation as observed with other Gram-negative bacteria. However, some <i>Brucella</i> strains interfere with the maturation of dendritic cells (DCs), which is essential for antigen-specific T-cell priming. In the present study, we investigated the interaction of human monocyte-derived DCs with the smooth attenuated <i>B. abortus</i> strain (S) 19, which has previously been employed successfully to vaccinate cattle.</p><p>Methodology/Principal findings</p><p>We first looked into the potential of S19 to hamper the cytokine-induced maturation of DCs; however, infected cells expressed CD25, CD40, CD80, and CD86 to a comparable extent as uninfected, cytokine-matured DCs. Furthermore, S19 activated DCs in the absence of exogeneous stimuli, enhanced the expression of HLA-ABC and HLA-DR, and was able to persist intracellularly without causing cytotoxicity. Thus, DCs provide a cellular niche for persisting brucellae <i>in vivo</i> as a permanent source of antigen. S19-infected DCs produced IL-12/23p40, IL-12p70, and IL-10, but not IL-23. While heat-killed bacteria also activated DCs, soluble mediators were not involved in S19-induced activation of human DCs. HEK 293 transfectants revealed cellular activation by S19 primarily through engagement of Toll-like receptor (TLR)2.</p><p>Conclusions/Significance</p><p>Thus, as an immunological prerequisite for vaccine efficacy, <i>B. abortus</i> S19 potently infects and potently activates (most likely via TLR2) human DCs to produce Th1-promoting cytokines.</p></div

Both <i>B.</i><i>abortus</i> S19 and strain 2308 induce IL-8 secretion by HEK293 cells overexpressing TLR2 but not TLR4.

<p>HEK293 cells were transiently transfected with human TLR2 (A) or TLR4 (B). After stimulation with LPS (rough, LPS Re595; smooth, LPS EH100) or heat-inactivated <i>B. abortus</i> S19 or strain 2308, NF-κB activation was assessed by IL-8 release by ELISA. Measurements were performed in triplicates, shown are mean values ± SD. One representative experiment out of two is shown.</p

<i>B.</i><i>abortus</i> S19 does not interfere with the cytokine-induced maturation of immature human DCs.

<p>Monocyte-derived immature DCs were infected with <i>B. abortus</i> S19 (MOI, 20) for 1 h, the bacteria were washed out, and the cells incubated in the presence of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, PGE₂). Uninfected control cells were incubated alongside in the presence or absence of cytokines. (A) After 48 h, the phenotype of the cells was determined by flow cytometry (bold lines, stimulated cells; grey areas, unstimulated cells; dotted lines, isotype controls). One representative of at least six independent experiments is shown. (B) Supernatants were analyzed for the presence of IL-12/23p40, IL-12p70, IL-23, and IL-10 (data expressed as median with interquartile range; n.s., not significant).</p

Prolonged intracellular survival of <i>B.</i><i>abortus</i> S19 in unstimulated DCs compared with cytokine-matured DCs.

<p>Immature DCs were infected with <i>B. abortus</i> S19 at MOI 20, the bacteria were washed out, antibiotics were added, and the cells were then incubated in the presence or absence or pro-inflammatory cytokines. Cells were lysed at day 0 to determine the initial number of intracellular bacteria, and on days 2, 6, and 10 to ascertain the intracellular bacterial numbers over time. CFUs differed significantly at d10 (*, p<0.05).</p

Increased MHC expression by strain S19-infected DCs.

<p>Immature DCs were infected with <i>B. abortus</i> S19 at MOI 20, the bacteria were washed out, antibiotics were added, and the cells were then incubated in the presence or absence or pro-inflammatory cytokines. After 42 h, the cellular expression of HLA-ABC (A) or HLA-DR (B) was determined by flow cytometry. Medians of the MFIs as well as the 25% and 75% percentiles of the MFIs for cells from eight donors (MFIs for isotype controls did not exceed 3.5). * p<0.05 compared to untreated DCs (Wilcoxon matched-pairs signed rank test).</p