Sensitive quantification of the HIV-1 reservoir in gut-associated lymphoid tissue
Moron-Lopez, Sara (Institut Germans Trias i Pujol. Institut de Recerca de la Sida IrsiCaixa)
Puertas, Maria C.. (Institut Germans Trias i Pujol. Institut de Recerca de la Sida IrsiCaixa)
Gálvez, Cristina (Institut Germans Trias i Pujol. Institut de Recerca de la Sida IrsiCaixa)
Navarro, Jordi (Hospital Universitari Vall d'Hebron)
Carrasco-Rozas, Ana (Hospital Universitari MútuaTerrassa (Terrassa, Catalunya))
Esteve, Maria (Hospital Universitari MútuaTerrassa (Terrassa, Catalunya))
Manyè, Josep (Institut Germans Trias i Pujol. Hospital Universitari Germans Trias i Pujol)
Crespo Casal, Manuel (Hospital Universitari Vall d'Hebron)
Salgado, Maria (Institut Germans Trias i Pujol. Institut de Recerca de la Sida IrsiCaixa)
Martinez Picado, Javier (Institut Germans Trias i Pujol. Institut de Recerca de la Sida IrsiCaixa)

Fecha:	2017
Resumen:	Background. The implementation of successful strategies to achieve an HIV cure has become a priority in HIV research. However, the current location and size of HIV reservoirs is still unknown since there are limited tools to evaluate HIV latency in viral sanctuaries such as gut-associated lymphoid tissue (GALT). As reported in the so called "Boston Patients", despite undetectable levels of proviral HIV-1 DNA in blood and GALT, viral rebound happens in just few months after ART interruption. This fact might imply that current methods are not sensitive enough to detect residual reservoirs. Showing that, it is imperative to improve the detection and quantification of HIV-1 reservoir in tissue samples. Herein, we propose a novel nonenzymatic protocol for purification of Lamina Propria Leukocytes (LPL) from gut biopsies combined to viral HIV DNA (vDNA) quantification by droplet digital PCR (ddPCR) to improve the sensitivity and accuracy of viral reservoir measurements (LPL-vDNA assay). Methods. Endoscopic ileum biopsies were sampled from 12 HIV-1-infected cART- suppressed subjects. We performed a DTT/EDTA-based treatment for epithelial layer removal followed by non-enzymatic disruption of the tissue to obtain lamina propria cell suspension (LP). CD45+ cells were subsequently purified by flow sorting and vDNA was determined by ddPCR. Results. vDNA quantification levels were significantly higher in purified LPLs (CD45+) than in bulk LPs (p<0. 01). The levels of vDNA were higher in ileum samples than in concurrent PBMC from the same individuals (p = 0. 002). As a result of the increased sensitivity of thispurification method, the Poisson 95% confidence intervals of the vDNA quantification data from LPLs were narrower than that from bulk LPs. Of note, vDNA was unambiguously quantified above the detection limit in 100% of LPL samples, while only in 58% of bulk LPs. Conclusion. We propose an innovative combined protocol for a more sensitive detection of the HIV reservoir in gut-associated viral sanctuaries, which might be used to evaluate any proposed eradication strategy.
Ayudas:	Ministerio de Economía y Competitividad FPDI-2013-17134 Agència de Gestió d'Ajuts Universitaris i de Recerca 2013FI_B 00275 Ministerio de Educación, Cultura y Deporte FPU15/03698
Nota:	Altres ajuts: This substudy was supported by ViiV and the American Foundation for AIDS Research(amfAR)(ARCHE). IrsiCaixa was supported by the CERCA programme from Generalitat de Catalunya.
Derechos:	Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, la comunicació pública de l'obra i la creació d'obres derivades, fins i tot amb finalitats comercials, sempre i quan es reconegui l'autoria de l'obra original.
Lengua:	Anglès
Documento:	Article ; recerca ; Versió publicada
Publicado en:	PloS one, Vol. 12 Núm. 4 (2017) , p. 1-10, ISSN 1932-6203

DOI: 10.1371/journal.pone.0175899
PMID: 28414780

10 p, 2.1 MB

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Documentos de investigación > Documentos de los grupos de investigación de la UAB > Centros y grupos de investigación (producción científica) > Ciencias de la salud y biociencias > Institut d'Investigació en Ciencies de la Salut Germans Trias i Pujol (IGTP)
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