title: Molecular methods for evaluation of virological status of nonhuman primates challenged with simian immunodeficiency or simian-human immunodeficiency viruses.
abstract: Nonhuman primates represent a robust model to evaluate preclinical efficacy of HIV-1 vaccine and therapeutic strategies. Plasma and tissue viral RNA as well as tissue proviral DNA load are key parameters in assessing efficacy of vaccines and therapeutics against simian immunodeficiency virus (SIV) or simian-human immunodeficiency virus (SHIV) challenge. To quantitate SIV RNA in plasma and tissues, an isothermal nucleic acid sequence-based amplification (NASBA) method using real-time detection of amplified RNA with molecular beacons was developed. This assay has accuracy and reproducibility over seven orders of magnitude and has advantages over the electrochemiluminescence-based NASBA assay described previously, both in terms of higher throughput and sensitivity. Reproducibility and accuracy were also demonstrated for a TaqMan real-time PCR assay for quantitating proviral DNA load in PBMCs and lymphoid tissues. In infected macaques, the level of plasma viremia correlated with the tissue viral RNA but not always with proviral DNA loads. Further, animals with undetectable levels of viral RNA in plasma and proviral DNA in tissues, showed no sign of seroconversion and activation of Gag-specific CD8+ or CD4+ T cells in peripheral blood. These results suggest that simultaneous application of real-time NASBA and PCR assays provides quantitative evaluation of challenge outcome in macaques.
authors.affiliation: Advanced BioScience Laboratories, Inc., Kensington, MD 20895, United States.
authors.forename: Eun Mi
authors.initials: EM
authors.lastname: Lee
authors.forename: Hye-Kyung
authors.initials: HK
authors.lastname: Chung
authors.forename: Jessica
authors.initials: J
authors.lastname: Livesay
authors.forename: John
authors.initials: J
authors.lastname: Suschak
authors.forename: Lindsay
authors.initials: L
authors.lastname: Finke
authors.forename: Lauren
authors.initials: L
authors.lastname: Hudacik
authors.forename: Lindsey
authors.initials: L
authors.lastname: Galmin
authors.forename: Britany
authors.initials: B
authors.lastname: Bowen
authors.forename: Phillip
authors.initials: P
authors.lastname: Markham
authors.forename: Anthony
authors.initials: A
authors.lastname: Cristillo
authors.forename: Ranajit
authors.initials: R
authors.lastname: Pal
mesh_terms: D000818:Animals; D004279:DNA, Viral / Q000032:analysis* / Q000235:genetics; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification*; D007963:Leukocytes, Mononuclear / Q000821:virology; D008221:Lymphoid Tissue / Q000821:virology; D008253:Macaca mulatta / Q000821:virology*; D010949:Plasma / Q000737:chemistry; D011533:Proviruses / Q000235:genetics / Q000302:isolation & purification; D012367:RNA, Viral / Q000032:analysis* / Q000235:genetics; D015203:Reproducibility of Results; D021161:Self-Sustained Sequence Replication / Q000379:methods; D012680:Sensitivity and Specificity; D015302:Simian Immunodeficiency Virus / Q000235:genetics / Q000302:isolation & purification*; D013696:Temperature; D019562:Viral Load / Q000379:methods*
chemical_list: D004279:DNA, Viral; D012367:RNA, Viral