Genetic instability of live, attenuated human immunodeficiency virus type 1 vaccine strains

Abstract

Live, attenuated viruses have been the most successful vaccines in monkey models of human immunodeficiency virus type 1 (HIV-1) infection. However, there are several safety concerns about using such an anti-HIV vaccine in humans, including reversion of the vaccine strain to virulence and recombination with endogenous retroviral sequences to produce new infectious and potentially pathogenic viruses. Because testing in humans would inevitably carry a substantial risk, we set out to test the genetic stability of multiply deleted HIV constructs in perpetuated tissue culture infections. The Delta3 candidate vaccine strain of HIV-1 contains deletions in the viral long terminal repeat (LTR) promoter and the vpr and nef genes. This virus replicates with delayed kinetics, but a profound enhancement of virus replication was observed after approximately 2 months of culturing. Analysis of the revertant viral genome indicated that the three introduced deletions were maintained but a 39-nucleotide sequence was inserted in the LTR promoter region. This insert was formed by duplication of the region encoding three binding sites for the Sp1 transcription factor. The duplicated Sp1 region was demonstrated to increase the LTR promoter activity, and a concomitant increase in the virus replication rate was measured. In fact, duplication of the Sp1 sites increased the fitness of the Delta3 virus (Vpr/Nef/U3) to levels higher than that of the singly deleted DeltaVpr virus. These results indicate that deleted HIV-1 vaccine strains can evolve into fast-replicating variants by multiplication of remaining sequence motifs, and their safety is therefore not guaranteed. This insight may guide future efforts to develop more stable anti-HIV vaccines.

FIG. 1

Improved growth kinetics of Δ3 revertant viruses. The HIV-1 samples represent the Δ3 virus after culturing on SupT1 cells for increasing periods. The perpetuated SupT1 infection was started by electroporation of 10 μg of Δ3 construct, and at the peak of infection, virus was passaged onto fresh, uninfected SupT1 cells. Supernatant samples were taken at several days posttransfection, frozen, and subsequently used to infect SupT1 cells with the same amount of input virus (1 ng CA-p24). Cell samples were also stored for proviral DNA analysis (see Fig. 2).

FIG. 2

The Δ3 mutant creates an LTR promoter with six Sp1 sites. (A) Cell samples taken on days 21, 28, 36, 42, 55, 73, 83, and 89 of the perpetuated SupT1 infection were used to extract cellular DNA as described previously (14), and the nef LTR region of the HIV-1 genome was PCR amplified. A 299-bp fragment is produced with the Δ3 mutant template (three Sp1 sites), and a revertant fragment of 338 bp is observed (six Sp1 sites). The day of cell harvest is indicated at the top of the gel. A 100-bp DNA ladder is provided in lanes 1 and 10 (lanes labeled M). (B) Quantitation of the ethidium bromide-stained gel was performed with the Kodak digital science 1D system and used to calculate the fractions of Δ3 mutant (three Sp1 sites) and Δ3 revertant (six Sp1 sites).

FIG. 3

Duplication of the three Sp1 sites in the LTR promoter. The wild-type LTR promoter contains two NF-κB sites (squares) at positions −105 to −96 and −91 to −82 relative to the RNA start site at +1 (arrow) and three Sp1 sites (circles) at positions −78 to −69, −67 to −58, and −56 to −47. The Δ3 LTR carries a deletion of the upstream part of the U3 promoter region (starting at position −150). The nucleotide sequence of the three Sp1 sites is shown, with the 10-mer binding sites underlined. The lower panel shows the Δ3 revertant with the 39-nt insert. The insert consists of a 32-nt duplication (arrows) and a 7-nt sequence of unknown origin (boxed). Of the three new Sp1 motifs, the upstream site III* is a partial copy of site III, and it is therefore unknown whether site III* can bind the Sp1 factor.

FIG. 4

The Δ3 LTR promoter gains activity by duplication of the Sp1 sites. (A) SupT1 T cells (5 × 10⁶) were electroporated with 40 μg of LTR-CAT reporter construct (wild type [open bars], Δ3 mutant [hatched bars], and Δ3 revertant [solid bars]) in the absence of Tat (left) or with 1 μg of LTR-CAT plasmid in combination with 2.5 μg of pcDNA3-Tat (middle). The cultures were harvested on day 3 for CAT assays. The fold Tat-mediated activation of LTR-transcription was calculated and is plotted (right). (B and C) Parallel transfections were performed on cells that were treated with PMA-PHA (final concentrations 25 ng/ml and 1 μg/ml, respectively) on day 1 posttransfection (B), and transfections were repeated in the presence of Sp1 expression plasmid pSVSp-1 (44a) (C). A representative experiment is shown, and similar results were obtained in four independent transfections. Furthermore, similar results were obtained in transfections with other cell types, including non-T cells. The basal and Tat-induced promoter activities cannot be compared directly because different amounts of LTR-CAT plasmid were used. When the results were corrected for this difference, an approximately 200-fold induction of LTR activity was measured.

FIG. 5

The reconstituted 6×Sp1 virus replicates with wild-type kinetics. Virus production in SupT1 cultures after infection with wild-type virus (▴) and the nef-U3 (■) and nef-U3-6×Sp1 (•) variants (A) and the vpr single-deletion mutant (▴), the vpr-nef-U3 (Δ3) mutant (■), and the vpr-nef-U3-6×Sp1 revertant (•) (B). The infections were performed in triplicate with different amounts of input virus: 0.2 ng of CA-p24 (top), 1.0 ng of CA-p24 (middle), and 5.0 ng of CA-p24 (bottom). Virus replication was monitored by measuring CA-p24 production in the culture supernatant. The cultures were split 1:5 at several times postinfection to sustain cell growth and virus replication; this resulted in small decreases in CA-p24 values.

FIG. 6

The 6×Sp1 variant does not improve replication in primary cells. PBMC cultures were infected with the wild-type virus and the nef-U3 and nef-U3-6×Sp1 variants (left) and the vpr single deletion mutant, the vpr-nef-U3 (Δ3) mutant, and the vpr-nef-U3-6×Sp1 revertant (right). Equal amounts of input virus was used (10 ng of CA-p24). Virus replication was monitored by measuring CA-p24 production in the culture supernatant.