title: Cross-clade ultrasensitive PCR-based assays to measure HIV persistence in large-cohort studies.
abstract: A small pool of infected cells persists in HIV-infected individuals receiving antiretroviral therapy (ART). Here, we developed ultrasensitive assays to precisely measure the frequency of cells harboring total HIV DNA, integrated HIV DNA, and two long terminal repeat (2-LTR) circles. These assays are performed on cell lysates, which circumvents the labor-intensive step of DNA extraction, and rely on the coquantification of each HIV molecular form together with CD3 gene sequences to precisely measure cell input. Using primary isolates from HIV subtypes A, B, C, D, and CRF01_A/E, we demonstrate that these assays can efficiently quantify low target copy numbers from diverse HIV subtypes. We further used these assays to measure total HIV DNA, integrated HIV DNA, and 2-LTR circles in CD4(+) T cells from HIV-infected subjects infected with subtype B. All samples obtained from ART-naive subjects were positive for the three HIV molecular forms (n = 15). Total HIV DNA, integrated HIV DNA, and 2-LTR circles were detected in, respectively, 100%, 94%, and 77% of the samples from individuals in which HIV was suppressed by ART. Higher levels of total HIV DNA and 2-LTR circles were detected in untreated subjects than individuals on ART (P = 0.0003 and P = 0.0004, respectively), while the frequency of CD4(+) T cells harboring integrated HIV DNA did not differ between the two groups. These results demonstrate that these novel assays have the ability to quantify very low levels of HIV DNA of multiple HIV subtypes without the need for nucleic acid extraction, making them well suited for the monitoring of viral persistence in large populations of HIV-infected individuals.
  
  
  OBJECTIVE
  Since the discovery of viral reservoirs in HIV-infected subjects receiving suppressive ART, measuring the degree of viral persistence has been one of the greatest challenges in the field of HIV research. Here, we report the development and validation of ultrasensitive assays to measure HIV persistence in HIV-infected individuals from multiple geographical regions. These assays are relatively inexpensive, do not require DNA extraction, and can be completed in a single day. Therefore, they are perfectly adapted to monitor HIV persistence in large cohorts of HIV-infected individuals and, given their sensitivity, can be used to monitor the efficacy of therapeutic strategies aimed at interfering with HIV persistence after prolonged ART.
authors.affiliation: Vaccine and Gene Therapy Institute Florida, Port St. Lucie, Florida, USA.
authors.forename: Claire
authors.initials: C
authors.lastname: Vandergeeten
authors.affiliation: Vaccine and Gene Therapy Institute Florida, Port St. Lucie, Florida, USA.
authors.forename: Rémi
authors.initials: R
authors.lastname: Fromentin
authors.affiliation: University of Milan, Clinic of Infectious Diseases, Department of Health Sciences, Milan, Italy.
authors.forename: Esther
authors.initials: E
authors.lastname: Merlini
authors.affiliation: Vaccine and Gene Therapy Institute Florida, Port St. Lucie, Florida, USA.
authors.forename: Mariam B
authors.initials: MB
authors.lastname: Lawani
authors.affiliation: Vaccine and Gene Therapy Institute Florida, Port St. Lucie, Florida, USA.
authors.forename: Sandrina
authors.initials: S
authors.lastname: DaFonseca
authors.affiliation: Vaccine and Gene Therapy Institute Florida, Port St. Lucie, Florida, USA.
authors.forename: Wendy
authors.initials: W
authors.lastname: Bakeman
authors.affiliation: Vaccine and Gene Therapy Institute Florida, Port St. Lucie, Florida, USA.
authors.forename: Amanda
authors.initials: A
authors.lastname: McNulty
authors.affiliation: Midway Immunology & Research Center, Fort Pierce, Florida, USA.
authors.forename: Moti
authors.initials: M
authors.lastname: Ramgopal
authors.affiliation: United States Military HIV Research Program, Silver Spring, Maryland, USA.
authors.forename: Nelson
authors.initials: N
authors.lastname: Michael
authors.affiliation: United States Military HIV Research Program, Silver Spring, Maryland, USA.
authors.forename: Jerome H
authors.initials: JH
authors.lastname: Kim
authors.affiliation: United States Military HIV Research Program, Silver Spring, Maryland, USA Thai Red Cross AIDS Research Centre, Pathumwan, Bangkok, Thailand.
authors.forename: Jintanat
authors.initials: J
authors.lastname: Ananworanich
authors.affiliation: Vaccine and Gene Therapy Institute Florida, Port St. Lucie, Florida, USA nchomont@vgtifl.org.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Chomont
mesh_terms: D015331:Cohort Studies; D004279:DNA, Viral / Q000032:analysis / Q000235:genetics; D006678:HIV / Q000235:genetics / Q000302:isolation & purification*; D015658:HIV Infections / Q000821:virology*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D019562:Viral Load / Q000379:methods*
chemical_list: D004279:DNA, Viral