9 research outputs found
Use of RT-defective HIV virions: new tool to evaluate specific response in chronic asymptomatic HIV-infected individuals
Background Generation of new reagents that can be used to screen or monitor HIV-1-specific responses constituted an interesting field in the development of HIV vaccines to improve their efficacy. Methods We have evaluated the specific T cell response against different types of NL4-3 virions (including NL4-3 aldrithiol-2 treated, NL4-3/ÎRT and R5 envelopes: NL4-3/ÎRT/ÎEnv[AC10] and NL4-3/ÎRT/ÎEnv[Bal]) and against pools of overlapping peptides (15 mer) encompassing the HIV-1 Gag and Nef regions. Cryopreserved PBMC from a subset of 69 chronic asymptomatic HIV positive individuals have been employed using different techniques including IFN-Îł ELISPOT assay, surface activation markers and intracellular cytokine staining (ICS) by flow cytometry. Results The differential response obtained against NL4-3 aldrithiol-2 treated and NL4-3/ÎRT virions (25% vs 55%, respectively) allow us to divide the population in three groups: "full-responders" (positive response against both viral particles), "partial-responders" (positive response only against NL4-3/ÎRT virions) and "non-responders" (negative responses). There was no difference between X4 and R5 envelopes. The magnitude of the total responses was higher against NL4-3/ÎRT and was positively correlated with gender and inverse correlated with viral load. On the contrary CD4+ T cell count was not associated with this response. In any case responses to the viruses tended to be lower in magnitude than those detected by the overlapping peptides tested. Finally we have found an increased frequency of HLA-B27 allele (23% vs 9%) and a significant reduction in some activation markers (CD69 and CD38) on T cells surface in responders vs non-responders individuals. Conclusions In summary these virions could be considered as alternative and useful reagents for screening HIV-1-specific T cell responses in HIV exposed uninfected people, HIV infected patients and to assess immunogenicity of new prototypes both in vitro and in vaccine trials, by a feasible, simply, effective and low cost assay
Use of RT-defective HIV virions: new tool to evaluate specific response in chronic asymptomatic HIV-infected individuals.
Background Generation of new reagents that can be used to screen or monitor HIV-1-specific responses constituted an interesting field in the development of HIV vaccines to improve their efficacy. Methods We have evaluated the specific T cell response against different types of NL4-3 virions (including NL4-3 aldrithiol-2 treated, NL4-3/ÎRT and R5 envelopes: NL4-3/ÎRT/ÎEnv[AC10] and NL4-3/ÎRT/ÎEnv[Bal]) and against pools of overlapping peptides (15 mer) encompassing the HIV-1 Gag and Nef regions. Cryopreserved PBMC from a subset of 69 chronic asymptomatic HIV positive individuals have been employed using different techniques including IFN-Îł ELISPOT assay, surface activation markers and intracellular cytokine staining (ICS) by flow cytometry. Results The differential response obtained against NL4-3 aldrithiol-2 treated and NL4-3/ÎRT virions (25% vs 55%, respectively) allow us to divide the population in three groups: "full-responders" (positive response against both viral particles), "partial-responders" (positive response only against NL4-3/ÎRT virions) and "non-responders" (negative responses). There was no difference between X4 and R5 envelopes. The magnitude of the total responses was higher against NL4-3/ÎRT and was positively correlated with gender and inverse correlated with viral load. On the contrary CD4+ T cell count was not associated with this response. In any case responses to the viruses tended to be lower in magnitude than those detected by the overlapping peptides tested. Finally we have found an increased frequency of HLA-B27 allele (23% vs 9%) and a significant reduction in some activation markers (CD69 and CD38) on T cells surface in responders vs non-responders individuals. Conclusions In summary these virions could be considered as alternative and useful reagents for screening HIV-1-specific T cell responses in HIV exposed uninfected people, HIV infected patients and to assess immunogenicity of new prototypes both in vitro and in vaccine trials, by a feasible, simply, effective and low cost assay
Use of RT-Defective HIV Virions: New Tool to Evaluate Specific Response in Chronic Asymptomatic HIV-Infected Individuals
<div><p>Background</p><p>Generation of new reagents that can be used to screen or monitor HIV-1-specific responses constituted an interesting field in the development of HIV vaccines to improve their efficacy.</p> <p>Methods</p><p>We have evaluated the specific T cell response against different types of NL4-3 virions (including NL4-3 aldrithiol-2 treated, NL4-3/ÎRT and R5 envelopes: NL4-3/ÎRT/ÎEnv[AC10] and NL4-3/ÎRT/ÎEnv[Bal]) and against pools of overlapping peptides (15 mer) encompassing the HIV-1 Gag and Nef regions. Cryopreserved PBMC from a subset of 69 chronic asymptomatic HIV positive individuals have been employed using different techniques including IFN-Îł ELISPOT assay, surface activation markers and intracellular cytokine staining (ICS) by flow cytometry.</p> <p>Results</p><p>The differential response obtained against NL4-3 aldrithiol-2 treated and NL4-3/ÎRT virions (25% vs 55%, respectively) allow us to divide the population in three groups: â<i>full-responders</i>â (positive response against both viral particles), <i>âpartial-respondersâ</i> (positive response only against NL4-3/ÎRT virions) and â<i>non-responders</i>â (negative responses). There was no difference between X4 and R5 envelopes. The magnitude of the total responses was higher against NL4-3/ÎRT and was positively correlated with gender and inverse correlated with viral load. On the contrary CD4+ T cell count was not associated with this response. In any case responses to the viruses tended to be lower in magnitude than those detected by the overlapping peptides tested. Finally we have found an increased frequency of HLA-B27 allele (23% vs 9%) and a significant reduction in some activation markers (CD69 and CD38) on T cells surface in responders vs non-responders individuals.</p> <p>Conclusions</p><p>In summary these virions could be considered as alternative and useful reagents for screening HIV-1-specific T cell responses in HIV exposed uninfected people, HIV infected patients and to assess immunogenicity of new prototypes both <i>in vitro</i> and in vaccine trials, by a feasible, simply, effective and low cost assay.</p> </div
Comparison of frequencies and magnitudes of responses against the different viral particles and peptides pools tested.
<p>(<b>A</b>) Bars show the percentage of individuals with positive response to the different constructs (Black bars) and the gag p24, p17 and small proteins (sp) and nef peptide pools (White bars) among the samples of HIV+ individuals tested. (<b>B</b>) Each symbol represents the mean of SFC (spot forming cells/10<sup>6</sup> cells) ± SEM counted per condition after background subtraction. <i>P</i> values were calculated using the <i>Wilcoxon Matched</i>-Pairs Ranks test for continuous variables and Ï2-test for categorical variables. The magnitude of response was significantly different among indicated stimuli (*p<0.05; **p<0.01; ***p<0.001).</p
Example of IFN-Îł response in CD4 and CD8 T cell subsets from different individuals: full responder (FR) vs non-responder (NR).
<p>(<b>A</b>) Representative dot plots of gating strategy used and cytokine responses detected using different stimuli. Box indicated the percentage of IFN-Îł+ T cells. PMA-Ionophore was used as a positive control. (<b>B</b>) Evaluation of cytokine production in both subsets (CD4 and CD8). Percentage of T cells producing IFN-Îł, TNF-α or IL-2 in response to virions (ÎRT or WT+AT2) or to a pool of peptides encompassing p24 is depicted.</p
Percentage of expression of activation markers (CD38 and CD69) in CD4 and CD8 T cell subsets.
<p>Extracellular staining revealed that baseline activation levels varied significantly between full-responders and non-responders HIV-infected individuals in CD4+CD38+ and CD4+CD69+ subsets. MannâWhitney P value is shown.</p
HLA-B subtypes carrier and allele frequencies in the asymptomatic population analyzed.
<p>HLA-B subtypes carrier and allele frequencies in the asymptomatic population analyzed.</p