Pilot studies for development of an HIV subtype panel for surveillance of global diversity

Abstract

The continued global spread and evolution of HIV diversity pose significant challenges to diagnostics and vaccine strategies. NIAID partnered with the FDA, WRAIR, academia, and industry to form a Viral Panel Working Group to design and prepare a panel of well-characterized current and diverse HIV isolates. Plasma samples that had screened positive for HIV infection and had evidence of recently acquired infection were donated by blood centers in North and South America, Europe, and Africa. A total of 80 plasma samples were tested by quantitative nucleic acid tests, p24 antigen, EIA, and Western blot to assign a Fiebig stage indicative of approximate time from initial infection. Evaluation of viral load using FDA-cleared assays showed excellent concordance when subtype B virus was tested, but lower correlations for subtype C. Plasma samples were cocultivated with phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMCs) from normal donors to generate 30 viral isolates (50-80% success rate for samples with viral load >10,000 copies/ml), which were then expanded to 10(7)-10(9) virus copies per ml. Analysis of env sequences showed that sequences derived from cultured PBMCs were not distinguishable from those obtained from the original plasma. The pilot collection includes 30 isolates representing subtypes B, C, B/F, CRF04_cpx, and CRF02_AG. These studies will serve as a basis for the development of a comprehensive panel of highly characterized viral isolates that reflects the current dynamic and complex HIV epidemic, and will be made available through the External Quality Assurance Program Oversight Laboratory (EQAPOL).

FIG. 1.

Culture expansion of virus. Virus from the initial HIV isolation was expanded in phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMCs) through two cycles of virus cultivation. The first cycle expanded the virus in T25 flasks (A) starting with 3×10⁶ infected PBMCs (open circles) or 1 ml of culture supernatant (closed squares) to 10 ml culture. The second cycle expanded these to 100 ml culture in T75 flasks (B). All cultures contained 3×10⁶ cells/ml of 3-day PHA-stimulated PBMCs from normal donors. The cultures were monitored daily for HIV p24 and harvested on day 11.

FIG. 2.

Relationship between HIV-1 viral load measurements of plasma samples from U.S. isolates (subtype B) or South African isolates (subtype C) as tested on Roche COBAS Ampliprep/COBAS TaqMan HIV-1 Test v2.0 vs. Roche COBAS Ampliprep/COBAS TaqMan HIV-1 Test v1.0, Roche Amplicor Monitor v1.5, Siemens bDNA v3.0, and the Abbott m2000 assays.

FIG. 3.

Comparison of viral sequences from plasma and derived cultured virus from a Fiebig stage II virus isolate (BP00002) from South Africa. The envelope sequence analysis of original plasma and virus cultures from supernatant or PBMCs of original isolate as described in Fig. 2 was carried out on 10–12 separate sequences of the respective virus preparations. Point mutations in each sequence are designated by a color marker to indicate specific mutation type (A). The observed mismatches mostly led to codon changes (B).

FIG. 4.

Subtype designation of virus isolates based on near-full-length RNA sequencing and phylogenetic analysis. The scale bar represents 1% genetic distance. Bootstrap values at relevant nodes are indicated. Reference sequences are shown alongside sequences derived from the viral panel (designated by a dot).