A dual infection/competition assay shows a correlation between ex vivo human immunodeficiency virus type 1 fitness and disease progression

Abstract

This study was designed to examine the impact of human immunodeficiency virus type 1 (HIV-1) fitness on disease progression through the use of a dual competition/heteroduplex tracking assay (HTA). Despite numerous studies on the impact of HIV-1 diversity and HIV-specific immune response on disease progression, we still do not have a firm understanding of the long-term pathogenesis of this virus. Strong and early CD8-positive cytotoxic T-cell and CD4-positive T-helper cell responses directed toward HIV-infected cells appear to curb HIV pathogenesis. However, the rate at which the virus infects the CD4(+) T-cell population and possibly destroys the HIV-specific immune response may also alter the rate of disease progression. For HIV-1 fitness studies, we established conditions for dual HIV-1 infections of peripheral blood mononuclear cells (PBMC) and a sensitive HTA to measure relative virus production. A pairwise comparison was then performed to estimate the relative fitness of various non-syncytium-inducing/CCR5-tropic (NSI/R5) and syncytium-inducing/CXCR4-tropic (SI/X4) HIV-1 isolates. Four HIV-1 strains (two NSI/R5 and two SI/X4) with moderate ex vivo fitness were then selected as controls and competed against primary HIV-1 isolates from an HIV-infected Belgian cohort. HIV-1 isolates from long-term survivors (LTS) were outcompeted by control strains and were significantly less fit than HIV-1 isolates from patients with accelerated progression to AIDS (PRO). In addition, NSI/R5 HIV-1 isolates from PRO overgrew control SI/X4 strains, suggesting that not all SI/X4 HIV-1 isolates replicate more efficiently than all NSI/R5 isolates. Finally, there were strong, independent correlations between viral load and the total relative fitness values of HIV-1 isolates from PRO (r = 0.84, P = 0.033) and LTS (r = 0.86, P = 0.028). Separation of the PRO and LTS plots suggest that HIV-1 fitness together with viral load may be a strong predictor for the rate of disease progression.

FIG. 1

Schematic representation of growth competition experiments and detection methods for dual infections. (A) Dual infections with a pair of HIV-1 isolates were performed at three different MOIs (wells II, III, and IV). Wells I and V correspond to positive controls for viruses 1 and 2. An uninfected culture (well VI) was used as a negative control. (B) Schema for PCR coamplification of two HIV-1 env fragments. Two env genomic fragments (C2-C4 and C2-V3 of the gp120-coding region) were PCR amplified from each dual (wells II, III, and IV) and single (wells I and V) HIV-1 infection, using first external primers (envB and ED14) and then one of two sets of nested primer pairs (E80-E105 or E80-E125). (C) Detection of two different HIV-1 isolates in growth competition experiments by HTA. Nested env PCR products from A-92UG029 and E-CMU06 competition were denatured and annealed to a subtype E probe (HIV-1 E-TH22). The percentage of each HIV-1 isolate in well III is shown below the autoradiograph. (D) Cloning/probe hybridization analysis of HIV-1 env fragments from a dual infection (well III). Ninety-six env bacterial colonies containing plasmid cloned from the 0.1:0.1-MOI dual infection (well III) were hybridized to subtype A or E env-specific oligonucleotides (4). The final percentage of each HIV-1 isolate in the dual infection is shown below the autoradiographs of hybridization blots.

FIG. 2

Growth competition experiment with HIV-1 isolates B-HXB2 and E-CMU06 to validate the use of HTA as a dual virus detection method. (A) Standard conditions used in this study to analyze final proportions of two HIV-1 isolates from a dual infection: (i) proviral DNA was extracted from infected cells, (ii) a 0.48-kb env fragment (C2-V3) was PCR amplified by an external/nested technique, and (iii) HTA was performed with a subtype-specific probe (e.g., E-TH22). The same conditions were used for the subsequent controls, except for a larger (0.66-kb) env PCR fragment (C2-V4) (B), a probe specific to the subtype of the other HIV-1 isolate in the competition (B-SF162) (C), or viral RNA from supernatant culture (D). Roman numbers above the lanes correspond with the dual infections in Fig. 1A. P, lane containing only the subtype-specific probe.

FIG. 3

Growth competition experiments and HTA controls with pairs of six primary HIV-1 isolates. Five pairs of two different SI/X4 HIV-1 isolates were used: (A) D-92UG021 plus F-93BR020, (B) D-92UG021 plus E-CMU06, (C) A-92UG029 plus D-93UG067, (D) A-92UG029 plus E-CMU06, and (E) B-HXB2 plus E-CMU06. The final percentage of each HIV-1 isolate in the dual infection was determined by HTA and by cloning/probe hybridization assays (indicated below well III). See legends to Fig. 1 and 2 for experimental details.

FIG. 4

Three growth competition experiments with NSI/R5 HIV-1 isolates. Production of two NSI/R5 isolates (A-92RW009 plus B-BaL, B-BaL plus C-92BR025, and A-92RW009 plus C-92BR025) in each competition was measured by HTA (A). Relative fitness values were then derived from the three NSI/R5 dual infections from each competition (B) as described for Table 3.

FIG. 5

The ex vivo fitness of HIV-1 isolated from LTS (A) and PRO (B) relative to four HIV-1 control strains. Within a 2- to 3-year period, two HIV-1 strains (designated A and B in the figure) were isolated from the three LTS and three PRO. A positive (red) or negative (blue) fitness difference corresponds to an lts or pro HIV-1 isolate being more or less fit than the HIV-1 control strain, respectively. Fitness differences were derived from the three dual infections in each competition with a control strain (two SI/X4 [A-92UG029 and E-CMU06] and two NSI/R5 [A-92RW009 and C-92BR025]). Limits of detection result in a maximum or minimum fitness difference of >100-fold. Dashed and solid bars correspond to competitions with SI/X4 and NSI/R5 patient HIV-1 isolates, respectively.

FIG. 6

Correlations between viral loads and the total relative fitness of HIV-1 isolates from infected individuals. Viral loads were plotted against the total relative fitness values of all HIV-1 isolates. Total relative fitness is the average of four relative fitness values, corresponding to the four competitions of each patient isolate with each of four control strains. Linear regression analysis (solid line) was then performed on the entire cohort (A), the LTS (B), and the PRO (B). Ninety-nine percent confidence curves (dashed lines) are shown in panel A.