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. 2013;8(3):e58927.
doi: 10.1371/journal.pone.0058927. Epub 2013 Mar 14.

Use of RT-defective HIV Virions: New Tool to Evaluate Specific Response in Chronic Asymptomatic HIV-infected Individuals

Free PMC article

Use of RT-defective HIV Virions: New Tool to Evaluate Specific Response in Chronic Asymptomatic HIV-infected Individuals

Alberto Crespo Guardo et al. PLoS One. .
Free PMC article


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/ΔRT and R5 envelopes: NL4-3/ΔRT/ΔEnv[AC10] and NL4-3/ΔRT/ΔEnv[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-γ 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/ΔRT 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/ΔRT 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/ΔRT 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.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.


Figure 1
Figure 1. Comparison of frequencies and magnitudes of responses against the different viral particles and peptides pools tested.
(A) 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) Each symbol represents the mean of SFC (spot forming cells/106 cells) ± SEM counted per condition after background subtraction. P values were calculated using the Wilcoxon Matched-Pairs Ranks test for continuous variables and χ2-test for categorical variables. The magnitude of response was significantly different among indicated stimuli (*p<0.05; **p<0.01; ***p<0.001).
Figure 2
Figure 2. Example of IFN-γ response in CD4 and CD8 T cell subsets from different individuals: full responder (FR) vs non-responder (NR).
(A) Representative dot plots of gating strategy used and cytokine responses detected using different stimuli. Box indicated the percentage of IFN-γ+ T cells. PMA-Ionophore was used as a positive control. (B) Evaluation of cytokine production in both subsets (CD4 and CD8). Percentage of T cells producing IFN-γ, TNF-α or IL-2 in response to virions (ΔRT or WT+AT2) or to a pool of peptides encompassing p24 is depicted.
Figure 3
Figure 3. Frequency of IFN-γ-ELISPOT positive responses against different pools of peptides.
(A) Population analyzed has been divided attending to their positive response against WT+AT2 and/or ΔRT immunogens into three different groups (FR: full-responders; PR: partial-responders and NR: non-responders). The response obtained against overlapping pools of peptides from Gag and Nef showed a different pattern between established groups. Gag response was predominant in responder individuals (see results). CEF peptide pool was used as an HIV-independent peptide stimulus. (B) Magnitude (SFC/106 PBMC) of HIV-1-specific T-cell responses against Gag (p24, p17 and p6-p7) protein was not affected among groups whereas Nef present a significant reduction in non-responder individuals. Epitopes from human cytomegalovirus, Epstein-Barr virus and influenza virus (CEF peptide pool) showed a higher magnitude in responder individuals. Statistically significant differences are shown (*p<0.05; **p<0.01; ***p<0.001).
Figure 4
Figure 4. Percentage of expression of activation markers (CD38 and CD69) in CD4 and CD8 T cell subsets.
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. Mann–Whitney P value is shown.

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Grant support

This study was partially supported by grants FIS PI040503, FIS PI070291, FIS PI1200969, RIS - Red Temática Cooperativa de Grupos de Investigación en Sida del Fondo de Investigación Sanitaria del Instituto de Salud Carlos III (ISCIII-RETIC RD06/0006), HIVACAT - Catalan Program for HIV Vaccine Development. Dr. Montserrat Plana is a researcher from the Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS) and is supported by the Spanish Health Institute Carlos III (ISCIII) and the Health Department of the Catalan Government (Generalitat de Catalunya). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.