Combination of quadruplex qPCR and next-generation sequencing for qualitative and quantitative analysis of the HIV-1 latent reservoir

Author:

Gaebler Christian1ORCID,Lorenzi Julio C.C.1,Oliveira Thiago Y.1,Nogueira Lilian1ORCID,Ramos Victor1ORCID,Lu Ching-Lan1,Pai Joy A.1ORCID,Mendoza Pilar1,Jankovic Mila1ORCID,Caskey Marina1,Nussenzweig Michel C.12ORCID

Affiliation:

1. Laboratory of Molecular Immunology, The Rockefeller University, New York, NY

2. Howard Hughes Medical Institute, The Rockefeller University, New York, NY

Abstract

HIV-1 infection requires lifelong therapy with antiretroviral drugs due to the existence of a latent reservoir of transcriptionally inactive integrated proviruses. The goal of HIV-1 cure research is to eliminate or functionally silence this reservoir. To this end, there are numerous ongoing studies to evaluate immunological approaches, including monoclonal antibody therapies. Evaluating the results of these studies requires sensitive and specific measures of the reservoir. Here, we describe a relatively high-throughput combined quantitative PCR (qPCR) and next-generation sequencing method. Four different qPCR probes covering the packaging signal (PS), group-specific antigen (gag), polymerase (pol), and envelope (env) are combined in a single multiplex reaction to detect the HIV-1 genome in limiting dilution samples followed by sequence verification of individual reactions that are positive for combinations of any two of the four probes (Q4PCR). This sensitive and specific approach allows for an unbiased characterization of the HIV-1 latent reservoir.

Funder

National Center for Advancing Translational Sciences

National Institutes of Health

Shapiro-Silverberg Fund

Bill and Melinda Gates Foundation

Einstein–Rockefeller–CUNY Center for AIDS Research

BEAT-HIV Delaney

Robertson Fund

Howard Hughes Medical Institute

Robert S. Wennett Post-Doctoral Fellowship

Publisher

Rockefeller University Press

Subject

Immunology,Immunology and Allergy

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