18 research outputs found

    SHIV-1157i and passaged progeny viruses encoding R5 HIV-1 clade C env cause AIDS in rhesus monkeys

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    Background: Infection of nonhuman primates with simian immunodeficiency virus (SIV) or chimeric simian-human immunodeficiency virus (SHIV) strains is widely used to study lentiviral pathogenesis, antiviral immunity and the efficacy of AIDS vaccine candidates. SHIV challenges allow assessment of anti-HIV-1 envelope responses in primates. As such, SHIVs should mimic natural HIV-1 infection in humans and, to address the pandemic, encode HIV-1 Env components representing major viral subtypes worldwide. Results: We have developed a panel of clade C R5-tropic SHIVs based upon env of a Zambian pediatric isolate of HIV-1 clade C, the world's most prevalent HIV-1 subtype. The parental infectious proviral clone, SHIV-1157i, was rapidly passaged through five rhesus monkeys. After AIDS developed in the first animal at week 123 post-inoculation, infected blood was infused into a sixth monkey. Virus reisolated at this late stage was still exclusively R5 tropic and mucosally transmissible. Here we describe the long-term follow-up of this initial cohort of six monkeys. Two have remained non-progressors, whereas the other four gradually progressed to AIDS within 123–270 weeks post-exposure. Two progressors succumbed to opportunistic infections, including a case of SV40 encephalitis. Conclusion: These data document the disease progression induced by the first mucosally transmissible, pathogenic R5 non-clade B SHIV and suggest that SHIV-1157i-derived viruses, including the late-stage, highly replication-competent SHIV-1157ipd3N4 previously described (Song et al., 2006), display biological characteristics that mirror those of HIV-1 clade C and support their expanded use for AIDS vaccine studies in nonhuman primates

    Live attenuated, nef-deleted SIV is pathogenic in most adult macaques after prolonged observation

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    OBJECTIVE: A live attenuated SIV vaccine strain, termed SIVmac239Delta3 and containing large deletions in, and the negative regulatory element, was previously shown to cause AIDS mostly in monkeys vaccinated as infants. In the present study, we demonstrate that SIVmac239Delta3 is pathogenic in most vaccinated adult monkeys, given enough time. METHODS: Eleven rhesus macaques vaccinated as adults with SIVmac239Delta3 were followed for extended periods (up to 6.8 years). RESULTS: We found signs of immune dysregulation in all 11 adult vaccinees. All animals developed persistently inverted CD4 : CD8 T-cell ratios, seven (64%) had persistent recurrent viremia, and six (55%) had decreased CD4 T-cell counts ( or = 10 copies/ml and cytoviremia was a poor prognostic sign. CONCLUSION: Our data demonstrate that with time, a live attenuated, multiply deleted SIV vaccine can cause immune dysregulation in most vaccine recipients, even in initially immune competent, healthy adults. Immune dysfunction can progress to full AIDS. However, pathogenic effects became evident only several years after vaccination. Thus, mass vaccination of humans with similarly constructed live attenuated HIV vaccines, recently suggested for countries with high HIV-1 transmission rates, seems contraindicated

    Quantitation of Simian Cytokine andβ-Chemokine mRNAs, using real-time reverse transcriptase-polymerase chain reaction: variations in expression during chronic primate lentivirus infection

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    Cytokines and β-chemokines are important mediators of the immune system and are expressed in many infectious diseases. To study cytokine and β-chemokine profiles during pathogenesis of lentiviral infection and progression to AIDS in rhesus macaques, we established new quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assays based on TaqMan chemistry. Using synthetic RNA standards, we quantified mRNAs of IL-2, IL-4, IL-6, IL-10, IL-12 p40, interferon γ (IFN-γ), tumor necrosis factor α (TNF-α), RANTES, macrophage inflammatory protein 1α (MIP-1α), and MIP-1β in unstimulated peripheral blood mononuclear cells (PBMCs) and lymph nodes from macaques chronically infected with SIV or SHIV. Viremic monkeys with decreased CD4+ T cell counts (<500 cells/μl) had significantly higher IL-10 mRNA expression than uninfected controls, which parallels the findings in HIV-1-infected humans. In addition, MIP-1α, MIP-1β, and RANTES mRNA expression increased in viremic monkeys with decreased CD4+ T cell counts; gene expression was inversely correlated with CD4+ T cell counts, but not viral load. The newly established quantitative real-time RT-PCR assays will allow the determination of cytokine and β-chemokine patterns in rhesus macaques in studies of microbial pathogenesis or vaccine development

    Detection of HIV-1 neutralizing antibodies in a human CD4⁺/CXCR4⁺/CCR5⁺ T-lymphoblastoid cell assay system.

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    Sensitive assays are needed to meaningfully assess low levels of neutralizing antibodies (NAbs) that may be important for protection against the acquisition of HIV-1 infection in vaccine recipients. The current assay of choice uses a non-lymphoid cell line (TZM-bl) that may lack sensitivity owing to over expression of CD4 and CCR5. We used transfection of a human CD4+/CXCR4+/α4β7+ T-lymphoblastoid cell line (A3.01) with a CMV IE promoter-driven CCR5neo vector to stably express CCR5. The resulting line, designated A3R5, is permissive to a wide range of CCR5-tropic circulating strains of HIV-1, including HIV-1 molecular clones containing a Tat-inducible Renilla luciferase reporter gene and expressing multiple Env subtypes. Flow cytometric analysis found CCR5 surface expression on A3R5 cells to be markedly less than TZM-bl but similar to CD3.8 stimulated PBMC. More importantly, neutralization mediated by a diverse panel of monoclonal antibodies, HIV-1 positive polyclonal sera and sCD4 was consistently greater in A3R5 compared to TZM-bl cells. The A3R5 cell line provides a novel approach to guide the development and qualification of promising new HIV-1 vaccine immunogens

    SHIV-1157i and passaged progeny viruses encoding R5 HIV-1 clade C env cause AIDS in rhesus monkeys

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    Abstract Background Infection of nonhuman primates with simian immunodeficiency virus (SIV) or chimeric simian-human immunodeficiency virus (SHIV) strains is widely used to study lentiviral pathogenesis, antiviral immunity and the efficacy of AIDS vaccine candidates. SHIV challenges allow assessment of anti-HIV-1 envelope responses in primates. As such, SHIVs should mimic natural HIV-1 infection in humans and, to address the pandemic, encode HIV-1 Env components representing major viral subtypes worldwide. Results We have developed a panel of clade C R5-tropic SHIVs based upon env of a Zambian pediatric isolate of HIV-1 clade C, the world's most prevalent HIV-1 subtype. The parental infectious proviral clone, SHIV-1157i, was rapidly passaged through five rhesus monkeys. After AIDS developed in the first animal at week 123 post-inoculation, infected blood was infused into a sixth monkey. Virus reisolated at this late stage was still exclusively R5 tropic and mucosally transmissible. Here we describe the long-term follow-up of this initial cohort of six monkeys. Two have remained non-progressors, whereas the other four gradually progressed to AIDS within 123–270 weeks post-exposure. Two progressors succumbed to opportunistic infections, including a case of SV40 encephalitis. Conclusion These data document the disease progression induced by the first mucosally transmissible, pathogenic R5 non-clade B SHIV and suggest that SHIV-1157i-derived viruses, including the late-stage, highly replication-competent SHIV-1157ipd3N4 previously described (Song et al., 2006), display biological characteristics that mirror those of HIV-1 clade C and support their expanded use for AIDS vaccine studies in nonhuman primates.</p

    Subtype B neutralization in A3R5 cells and TZM-bl.

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    <p><b>A</b>-<b>C</b>. <b>Neutralization sensitivity of three tier 2 subtype B IMC.LucR transmitted/founder viruses to sCD4 and a panel of four mAbs in two A3R5 cell lines and TZM-bl</b>. With the exception where no inhibition was noted for any cell line, the A3R5 cell lines showed greater neutralization for all inhibitors tested compared to TZM-bl. <b>D</b>. <b>Grouped inhibitor comparison between cell lines</b>. When grouped by cell line using the non-parametric Mann-Whitney test, there was a significant difference between the A3R5 cell lines and TZM-bl (Mann-Whitney test; p<0.001). IC50 titers are the concentration of inhibitor at which the Renilla luciferase signal (RLUs) was reduced by 50% compared to the virus control (no inhibitor). Inhibitor concentrations ranged from 0.78 μg/mL to 25 μg/mL. All assays were performed in the presence of DEAE-Dextran.</p

    Initial characterization of A3R5 cell lines.

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    <p><b>A. CCR5 surface receptor number in sequentially sorted A3R5 cell lines.</b> For each sort, 2.0 x 10<sup>7</sup> cells were subjected to fluorescence activated cell sorting with the brightest population (top 25%) selected for expansion. Receptor number was calculated using Simply Cellular Beads. In general, each successive sort resulted in increasing CCR5 intensity on the cell surface. <b>B. Titration of NL.LucR-env.ecto in A3R5.6, TZM-bl and PHA/IL2 PBMC.</b> Replication competent reporter viruses encoding subtype B env (NL-LucR.T2A-SF162.ecto, NL-LucR.T2A-BaL.ecto) and subtype AE env (NL-LucR.T2A-CM235.ecto) were titered in each target cell. Background luciferase activity (cell control wells) was subtracted prior to plotting. The data show similar virus growth in the three target cells. <b>C. Infectivity of IMC.LucR viruses from multiple subtypes in various CCR5-expressing cell lines.</b> IMC.LucR from subtypes B, A/E and C were screened at a 1:3 dilution in triplicate for their ability to infect four different sorts of A3R5 cells as well as the SupT1.CCR5 and CEM.NKR.CCR5.Luc cell lines. The average relative light unit (RLU) values are indicated. IMC described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077756#pone.0077756.s002" target="_blank">Table S1</a>. While all cell lines supported IMC.LucR replication, infection was determined to be optimal in the A3R5.7 cell line. <b>D. Titration of IMC.LucR in A3R5.6 and A3R5.7 cells.</b> Replication competent reporter viruses encoding subtype B Env IMC (NL.LucR-SF162.ecto and NL.LucR-BaL.ecto) and subtype CRF_01 AE Env IMC (CM235, C1080, 0503M02138 and R2184 with backbone: NL.LucR-env.ecto) and subtype C Env IMC (ETH2220.LucR-ETH2220) were titered in the presence of DEAE-Dextran. Virus replication was measured as relative luminescence units (RLUs). Background luciferase activity (cell control wells) was similar in both cell lines and subtracted prior to plotting. Data represents the mean of three replicates with standard error bars (SEM). The data indicate no significant difference in IMC.LucR growth between the A3R5.6 and A3R5.7 cell lines (paired t-test;p0.5).</p

    Comparison of neutralization sensitivity in A3R5 and TZM-bl cell lines.

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    <p>A panel of eleven HIV+ CRF_01 AE sera was assayed against two tier 1 (C3347.c11 and C1080.c03) and four tier 2 (CM235-2, R2184.c04, 254003P00Ra.1 and 703357.c02) CRR_01 AE Env.IMC.LucR viruses in the A3R5.6 and TZM-bl cell lines. ID50 titers are the dilution of serum at which the Renilla luciferase signal was reduced by 50% compared to the virus control (no serum). Neutralization sensitivity was significantly greater (p<0.001; Wilcoxon Rank Sum) in the A3R5 cell line compared to TZM-bl for all virus/sera pairs tested. Similar results were obtained with the A3R5.7 cell line.</p

    Receptor expression in A3R5.7 cells.

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    <p><b>A. Flow cytometric analysis of CD4, CCR5 and α<sub>4</sub>β<sub>7</sub> expression in the A3R5.7 cell line.</b> 0.5 x 10<sup>6</sup> cells were singly stained for 30 minutes with fluorochrome-conjugated antibodies as shown followed by fixation in 2% paraformaldehyde. Data are representative of at least two independent experiments. Isotype controls are shown in grey. Nearly all cells were positive for CD4 and CCR5 while approximately half were positive for α<sub>4</sub>β<sub>7</sub>. <b>B. Comparison of cell surface CD4, CCR5 and α<sub>4</sub>β<sub>7</sub> receptor densities in various cell targets.</b> 0.5 x 10<sup>6</sup> cells were stained with fluorochrome-conjugated antibodies and compared to defined populations of similarly stained Quantum Simply Cellular beads. PBMC were stimulated with CD3.8 bi-specific antibody in the presence of 50U/mL rhIL-2. Assuming monovalent antibody-to-surface receptor binding, the Antibody Binding Capacity (ABC) calculated is equivalent to receptors/cell. Data represents the mean of two separate experiments. TZM-bl cells express high levels of CD4 and CCR5 but are negative for α<sub>4</sub>β<sub>7</sub> while A3R5.7 cells possess CCR5 and α<sub>4</sub>β<sub>7</sub> densities more similar to PBMC. CD4 expression on TZM-bl was beyond assay range. </p
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