HIV sequence variation associated with env antisense adoptive T-cell therapy in the hNSG mouse model

Abstract

The first use of lentiviral vectors in humans involved transduction of mature T-cells with an human immunodeficiency virus (HIV)-derived env antisense (envAS) vector to protect cells from HIV infection. In that study, only a minority of the patient T-cell population could be gene-modified, raising the question of whether the altered cells could affect replicating HIV populations. We investigated this using humanized NOD/SCID IL-2Rgamma(null) (hNSG) mice reconstituted with approximately 4-11% envAS-modified human T-cells. Mice were challenged with HIV-1(NL4-3), which has an env perfectly complementary to envAS, or with HIV-1(BaL), which has a divergent env. No differences were seen in viral titer between mice that received envAS-modified cells and control mice that did not. Using 454/Roche pyrosequencing, we analyzed the mutational spectrum in HIV populations in serum-from 33 mice we recovered 84,074 total reads comprising 31,290 unique sequence variants. We found enrichment of A-to-G transitions and deletions in envAS-treated mice, paralleling a previous tissue culture study where most target cells contained envAS, even though minority of cells were envAS-modified here. Unexpectedly, this enrichment was only detected after the challenge with HIV-1(BaL), where the viral genome would form an imperfect duplex with envAS, and not HIV-1(NL4-3), where a perfectly matched duplex would form.

Figure 1

Analysis of hNSG mice with ~4–11% VRX494 envAS-vector modified T-cells and challenged with HIV_NL4-3 or HIV_BaL. (a) The VRX494 vector. (b) Time line of mouse transplantation and infection. The hNSG mice were transplanted on day 0, then challenged with HIV_NL4-3 or HIV_BaL on day 30. (c) Flow cytometry analysis of transduction of CD4⁺ T-cells with envAS. Cells with high transduction levels were obtained as evidenced by GFP detection. (d) Analysis of the levels of T-cell engraftment. (e) Analysis of the levels of engraftment of VRX494-transduced cells, analyzed by sorting GFP⁺ cells. (f) Analysis of the level of HIV p24 antigen in serum at 48 days after infection. cPPT, central polypurine tract; CTS, central termination sequence; envAS, env antisense; GFP, green fluorescent protein; HIV, human immunodeficiency virus; hNSG, humanized NOD/SCID IL-2Rγ^null; LTR, long terminal repeat; RRE, rev-responsive element.

Figure 2

The HIV_NL4-3 genome, showing the regions targeted by the VRX494 envAS, and the HIV env amplicons used in this study. The numbering in the env gene refers to the HIV_NL4-3 genome. The envAS-targeted region extends from 6,601–7,538 (see blow-up). Three amplicons were designed to recover potential deletions and A→G changes. The relative positions and length of the amplicons (including 454 adapters and barcodes) are shown. Three amplicons were similarly designed in the homologous region of the HIV_BaL genome. envAS, env antisense; HIV, human immunodeficiency virus.

Figure 3

Box plots illustrating the types of base substitutions that accumulated during growth of HIV-1 in hNSG mice. (a) Base substitutions that accumulated during growth of HIV_BaL in the vector-treated and control mice. (b) Base substitutions that accumulated during growth of HIV_NL4-3 in the vector-treated and control mice. The boxes comprise all sequences with proportion changes in the middle two quartiles. Outliers, defined as sequences beyond 1.5 times the interquartile range, are plotted as open black circles. HIV, human immunodeficiency virus; hNSG, humanized NOD/SCID IL-2Rγ^null.

Figure 4

Comparison of the 100 sequences with the greatest enrichment of A–G transitions from the VRX494-treated and control mice challenged with HIV_BaL. The most enriched sequences from viruses grown in vector-treated mice (top) are compared to the most enriched sequences from controls (bottom). The bases were color-coded as indicated at the bottom of the figure. Only base positions that were A (in yellow) in the starting viral stock are shown, those substituted with G are shown in red. Grey indicates sequence gaps.

Figure 5

Statistical analysis of base substitution frequencies in vector-treated and control mice. For the statistical analysis, each mouse was treated as an individual measure of proportions. (a) Comparison of vector-treated and control mice after HIV_BaL challenge. (b) Comparison of vector-treated and control mice after HIV_NL4-3 challenge. In each panel, the x axis indicates the extent of enrichment per sequence for each base substitution used in the analysis, so that at any indicated enrichment score, only sequences with at least that score were considered. Progressing from left to right indicates analysis of increasingly high levels of substitution. The y axis indicates the –log₁₀ P value from the Mann–Whitney nonparametric comparison of means (one-sided) for the excess in vector-treated cohort compared to control cohort. The analysis was carried out for each of the 12 base substitutions. The horizontal red line indicates the threshold for achieving statistical significance at P values ≤0.05 (above is significant). (c) Scatter plot showing the proportions of sequences with enrichment scores >1.3 (P values <0.05) of A–G transitions for each mouse in the HIV_BaL group. HIV, human immunodeficiency virus.

Figure 6

Frequency of deletions in HIV-1 challenge viruses grown in the presence of vector-treated cells or controls. (a) Illustration of the numbers and locations of deletions in HIV_BaL from the vector-treated and control groups corresponding to the 5′-end of amplicon 2. Gray indicates sequence gaps. Deletions of ≥70 bases were plotted. (b) Analysis of the significance of the difference in deletion frequencies between vector-treated and control mice after HIV_BaL challenge. The x axis shows the length of deletions included in the analysis, so that at any indicated value only deletions of that length or greater were included in the analysis. The y axis shows the P value for the comparison of means between vector-treated and control groups calculated using the nonparametric Mann–Whitney test (one-sided). Each mouse was treated as a single measurement of proportions. (c) As in b, but analysis of the HIV_NL4-3 challenge group. (d) Comparison of proportions for deletions ≥70 bases, for the control and vector-treated mice. Each mouse is shown as a point.