Evolution of broadly cross-reactive HIV-1-neutralizing activity: therapy-associated decline, positive association with detectable viremia, and partial restoration of B-cell subpopulations

Abstract

Little is known about the stability of HIV-1 cross-neutralizing responses. Taking into account the fact that neutralization breadth has been positively associated with plasma viral load, there is no explanation for the presence of broadly neutralizing responses in a group of patients on treatment with undetectable viremia. In addition, the B-cell profile responsible for broadly cross-neutralizing responses is unknown. Here we studied the evolution of neutralizing responses and the B-cell subpopulation distribution in a group of patients with broadly cross-reactive HIV-1-neutralizing activity. We studied neutralization breadth evolution in a group of six previously identified broadly cross-neutralizing patients and six control patients during a 6-year period with a previously described minipanel of recombinant viruses from five different subtypes. B-cell subpopulation distribution during the study was also determined by multiparametric flow cytometry. Broadly cross-neutralizing activity was transient in four broad cross-neutralizers and stable, up to 4.6 years, in the other two. In four out of five broad cross-neutralizers who initiated treatment, a neutralization breadth loss occurred after viremia had been suppressed for as much as 20 months. B-cell subpopulation analyses revealed a significant increase in the frequency of naive B cells in broadly cross-reactive samples, compared with samples with less neutralization breadth (increased from 44% to 62%). We also observed a significant decrease in tissue-like and activated memory B cells (decreased from 19% to 12% and from 17% to 9%, respectively). Our data suggest that HIV-1 broadly cross-neutralizing activity is variable over time and associated with detectable viremia and partial B-cell restoration.

Fig 1

Study chart. Time zero corresponds to the last time point analyzed in the previous study (15). The period included in the present study before and after the previous cross-sectional study (mean and range) is indicated. The periods in which some patients were on cART are also indicated.

Fig 2

Changes in neutralization breadth, CD4⁺ T cells, and viral loads in the bCrN group. Neutralization breadth values (shown by the gray area and open circles) indicate the number of viruses from different subtypes neutralized out of a previously described minipanel (15). The number of CD4⁺ T cells/mm³ (CD4⁺ abs) is represented by a solid red line. The viral loads [log(VL), in copies/ml] are represented by a dotted blue line. The periods of time in which some patients were on cART are indicated. Time zero corresponds to the last time point included in the previous study (15). Antibodies capable of neutralizing across 5 subtypes were detected in our cohort of bCrN patients for a period up to 4.6 years. Four out of five bCrN patients that initiated cART showed a loss of neutralization breadth over time.

Fig 3

Changes in neutralization breadth, CD4⁺ T cells, and viral loads in the patient control group. Neutralization breadth values (shown by the gray area and open circles) indicate the number of viruses from different subtypes neutralized out of a previously described minipanel (15). The number of CD4⁺ T cells/mm³ (CD4⁺ abs) is represented by a solid red line. The viral loads [log(VL), in copies/ml] are represented by a dotted blue line. The periods of time in which some patients were on cART are indicated. Time zero corresponds to the last time point included in the previous study (15).

Fig 4

Breadth of the plasma NAb responses and plasma viral load (VL) levels. (A) Analysis of the overall neutralization breadth for plasma samples from patients with detectable (Det.) and undetectable (Und.) viremia (bCrN and control patients). (B) Comparison of neutralization breadth in plasma samples from patients with at least 6 months of undetectable viremia with the group of plasma samples from viremic patients and patients with less than 6 months of undetectable viremia. Horizontal bars within the point plots indicate the median subtype crossed ± the standard error of the mean (SEM). Significance between groups is indicated above the groups. Mann-Whitney U tests were used for comparisons between groups. Simple comparisons were made with use of a two-sided alpha level of 0.05.

Fig 5

Distribution of B-cell subpopulations before and after cART. The percentages of cells in each of the six B-cell subpopulations for samples from patients before and after cART are shown. The mean frequencies for different B-cell subpopulations are denoted by different colors. P values from comparisons of B-cell subpopulations are also shown. NS, not significant.

Fig 6

Distribution of B-cell subpopulations in samples with different neutralization breadths. (A) The percentages of cells in each of the six B-cell subpopulations were measured for samples from patients capable of neutralizing viruses across 4 or more subtypes and patients that neutralized viruses from fewer than 4 subtypes. The mean frequencies for different B-cell subpopulations are denoted by different colors. (B) Statistical analysis of each B-cell subpopulation. Horizontal bars within the point plots indicate the median percentage for each group ± the SEM. Significance between groups is indicated above the groups. Each type of symbol corresponds to a different patient. Black symbols, control patients; green diamonds, patient 181; red squares, patient 308; blue circles, patient 363; pink stars, patient 488; yellow triangles, patient 528; and orange inverted triangles, patient 541. Mann-Whitney U tests were used for comparisons between groups. Simple comparisons were made with use of a two-sided alpha level of 0.05.