Variants from the diverse virus population identified at seroconversion of a clade A human immunodeficiency virus type 1-infected woman have distinct biological properties

Abstract

Development of effective therapeutics to prevent new infections with human immunodeficiency type 1 (HIV-1) is predicated on an understanding of the properties that provide a selective advantage to a transmitted viral population. In contrast to the homogeneous virus population that typifies early HIV-1 infection of men, the viral population in women recently infected with clade A HIV-1 is genetically diverse, based on evaluation of the envelope gene. A longitudinal study of viral envelope evolution in several women suggested that representative envelope variants detected at seroconversion had distinct biological properties that affected viral fitness. To test this hypothesis, a full-length, infectious molecular clone, Q23-17, was obtained from an infected woman 1 year following seroconversion, and chimeric viruses containing envelope genes representative of seroconversion and 27-month-postseroconversion populations were constructed. Dendritic cells (DC) could transfer infection of seroconversion variant Q23ScA, which dominated the viral population in the year following seroconversion, and the closely related 1-year isolate Q23-17 to resting peripheral blood mononuclear cells (PBMC). In contrast, resting PBMC exposed to DC pulsed with Q23ScB, which was detected infrequently in samples after seroconversion, or the 27-month chimeras were inconsistently infected. Additionally, quiescent PBMC infected with Q23ScA or Q23-17 proliferated more robustly than uninfected cells or cells infected with the other envelope chimeras in response to immobilized anti-CD3. Stimulation with tetanus toxoid led to an increased proportion of CD45RA+ cells and a decreased expression of CD28 on CD45RO+ cells in cultures of Q23-17-infected PBMC. These data demonstrate that variants from the heterogeneous seroconversion clade A HIV-1 population in a Kenyan woman have distinct biological features that may influence viral pathogenesis.

FIG. 1

Restriction site map of the 3′ subclone of Q23-17 used for Q23 envelope chimera construction. Restriction sites used in chimera construction are shown above the scale, and the positions of redundant sites are given below. The HindIII site in the LTR that was used to clone Q23-17 is shown. XhoI is located in the vector. The positions of gp120 (SU) and gp41 (TM) are indicated. The gene encoding gp120 is closely flanked by NdeI and EcoNI sites.

FIG. 2

Changes in genetic diversity in envelope sequences sampled from subject Q23 during the 27 months following detection of seroconversion. Sequences used in this analysis are described in reference . (A) Percentage of Q23ScgA and Q23ScgB sequences detected in the total virus population at each sample point. Time is indicated in months from the seroconversion sample. Nucleotide sequences of variants within each group differed, on average, by 1.1% from other sequences in the group. For the first 17 months, all sequences could be classified as Q23ScgA or Q23ScgB. At 17 months postseroconversion (indicated by the arrow), sequences containing V1 insertions were first noted. After this time point, Q23ScgA or Q23ScgB sequences were infrequently detected. (B) Percent average pairwise distance of all sequences detected at each sample point. Error bars show standard deviations. Time points where envelope sequences were recovered that were used to construct viral chimeras described in this report are indicated.

FIG. 3

Sequence comparison of Q23 envelope chimeric viruses. Sequences for the region of the envelope gene spanning the NdeI and EcoNI sites are shown in single amino acid code. Regions of sequence homology are indicated by dots. At positions where sequences differ, the nonhomologous amino acid is shown. Potential N-linked glycosylation sites are underlined. Arrows indicate positions where the two seroconversion sequences have a common amino acid and the two 27-month sequences share a different amino acid. Clone Q23-17, which was isolated at an intermediate time point, is similar to either seroconversion or 27-month sequences at these positions.

FIG. 4

Response of PBMC infected with Q23 envelope chimeric viruses to immobilized CD3 antibody. PBMC were isolated from whole blood, stimulated with PHA, and infected as described in the text. Cells were rested for 3 to 5 days in the absence of IL-2, and 5 × 10⁴ cells were transferred to wells containing immobilized antibody to CD3 or IgG. After 5 days, plates were monitored for cell proliferation by the MTT assay. Results are shown as the mean and standard error of the SI (SI = OD_CD3/OD_IgG) for each of four replicate experiments. With the colorimetric assay, the maximum SI in these experiments was 2.7 (see Materials and Methods). Statistical significance between virus-infected cells and uninfected cells was determined by Student’s t test and is indicated by ∗ (P < 0.05). Cells infected with Q23ScA proliferated significantly better than did Q23LD-infected cells (P < 0.05), but the proliferative difference between Q23ScA-infected and uninfected cells was not highly significant (P < 0.09).

FIG. 5

Phenotypic changes in cells infected with Q23-17 in response to stimulation with TT. Data shown are representative of the three replicate experiments summarized in Table 3. PBMC were PHA stimulated, infected at an MOI of 0.0001, and rested for 5 days as described in the text. DC were pulsed with TT and added to autologous infected PBMC at a ratio of 1:100. Following an additional 5 days of culture, cells were collected, washed, stained with monoclonal antibodies to CD45RA or CD45RO (phycoerythrin conjugated) and CD28 or CD25 (fluorescein isothiocyanate conjugated), and evaluated by flow cytometry. Numbers in the diagrams indicate the proportion of positive cells in each quadrant; the proportion of double-positive cells is indicated in the upper right quadrant.