Selection pressure dynamics for all time points of the three lineages.

<p>(A) Selection pressure changes of the VRC26 lineage heavy (left) and light (right) chains. (B) Selection pressure changes of the CH103 lineage heavy (left) and light (right) chains. (C) Selection pressure changes of the VRC01 clade 03+06 heavy (left) and light (right) chains. (D) Selection pressure changes of the VRC01 clade 08 heavy (left) and light (right) chains. (E) Selection pressure changes of the VRC01 clade H3. (F) Selection pressure changes of the VRC01 clade L3. The selection strength for each time point of a lineage chain was measured using BASELINe. The same datasets used for evolutionary rate calculation were used to calculate selection strength. The mean and 95% HPD interval of selection strength for the CDRs (magenta) and FWRs (blue) were calculated separately. The statistical significance of the measured selection strength is shown on the bottom of the plot with ‘-’, ‘+’ and ‘n’ denoting negative selection, positive selection, and neutral selection, respectively.</p

The longitudinal changes of the predicted mutability correlate weakly with evolutionary rate decrease.

<p>(A) Predicted mutability for the stages of the three lineages. (B) The correlations between predicted mutability and evolutionary rate of CDRs were estimated using linear regression. No statistically significant correlation between the selection strength and evolutionary rate was observed. (C) The linear correlations between predicted mutability and evolutionary rate of FWRs. No significant correlation was observed for the three lineages.</p

Effects of Darwinian Selection and Mutability on Rate of Broadly Neutralizing Antibody Evolution during HIV-1 Infection

<div><p>Accumulation of somatic mutations in antibody variable regions is critical for antibody affinity maturation, with HIV-1 broadly neutralizing antibodies (bnAbs) generally requiring years to develop. We recently found that the rate at which mutations accumulate decreases over time, but the mechanism governing this slowing is unclear. In this study, we investigated whether natural selection and/or mutability of the antibody variable region contributed significantly to observed decrease in rate. We used longitudinally sampled sequences of immunoglobulin transcripts of single lineages from each of 3 donors, as determined by next generation sequencing. We estimated the evolutionary rates of the complementarity determining regions (CDRs), which are most significant for functional selection, and found they evolved about 1.5- to 2- fold faster than the framework regions. We also analyzed the presence of AID hotspots and coldspots at different points in lineage development and observed an average decrease in mutability of less than 10 percent over time. Altogether, the correlation between Darwinian selection strength and evolutionary rate trended toward significance, especially for CDRs, but cannot fully explain the observed changes in evolutionary rate. The mutability modulated by AID hotspots and coldspots changes correlated only weakly with evolutionary rates. The combined effects of Darwinian selection and mutability contribute substantially to, but do not fully explain, evolutionary rate change for HIV-1-targeting bnAb lineages.</p></div

Selection pressure changes of the three lineages correlate with evolutionary rate decrease.

<p>(A) Estimated selection strength for the stages of the three lineages. For all three lineages, the negative selection strength of later stages is comparable or stronger than earlier stages. The statistical significance of the measured selection strength is shown on the bottom of the plot with ‘-’, ‘+’ and ‘n’ denoting negative selection, positive selection, and neutral selection, respectively. (B) The selection strength on CDRs showed trends of correlation with the slowing of the evolutionary rate. Only the linear correlation for the CDRs of the VRC01 lineage is statistically significant. (C) The correlations between selection strength and evolutionary rate of FWRs. There are trends of correlation but none is statistically significant.</p

The evolutionary rates of the three antibody lineages decrease over time.

<p>The evolutionary rates for the stages of the three lineages were calculated using BEAST2. The mean evolutionary rates for the VRC26, CH103, and VRC01 lineages decreased about 80%, 45%, and 35% respectively over their study periods. However, the observed evolutionary rate decrease is not statistically significant for clade 08 light chain, clade H3, and L3. Each stage is labeled with the format of lineage name, chain name, and stage. Because clades of the VRC01 lineage showed more than 30% divergence from each other, we chose four representative clades (03+06, 08, H3, and L3) and calculated their evolutionary rates separately. The mean and 95% HPD are shown for each stage. Stages from the same chain of a lineage are connected to reveal longitudinal trends. The evolutionary rates of heavy chains and light chains are colored blue and red respectively.</p

The stage method is better than the time-bin method for estimating evolutionary rate dynamics.

<p>(A) The evolutionary rate for the constant-rate (CR) dataset was estimated using both restricted and relaxed log-normal clock models (labeled CR_restricted_clock and CR_lognormal_clock respectively). The estimated mean evolutionary rates from the two models are in good agreement. The mean evolutionary rates of the decreasing rate (DR) dataset estimated from the MCC tree and the 1000 time scaled Bayesian trees (labeled DR_MCC_tree and DR_1000_tree respectively) are highly consistent with the expected rates (red dots) derived from the calculated evolutionary rate of the CR dataset (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004940#sec012" target="_blank">Materials and Methods</a>). (B) The mean evolutionary rates for the eight time bins of the DR dataset were estimated from a single MCC tree (blue) and the mean and the 95% highest probability density (HPD) intervals estimated from 1000 time scaled Bayesian trees (green). The estimated evolutionary rates for each bin are significantly different from the expected rate (red). (C) The expected rates (red) of the three stages of the DR dataset are within the estimated 95% HPD of the mean evolutionary rates (purple), suggesting the stage method is reliable for characterizing evolutionary rate changes over time.</p

Dynamics of the predicted mutability for all time points of the three lineages.

<p>(A) Changes of predicted mutability of the VRC26 heavy (left) and light (right) chains. (B) Changes of predicted mutability of the CH103 heavy (left) and light (right) chains. (C) Changes of predicted mutability of the VRC01 clade 03+06 heavy (left) and light (right) chains. (D) Changes of predicted mutability of the VRC01 clade 08 heavy (left) and light (right) chains. (E) Changes of predicted mutability of the VRC01 clade H3. (F) Changes of predicted mutability of the VRC01 clade L3. Dashed line represents the mutability of UCA (VRC26 and CH103) or MRCA (VRC01 clades) of the lineages.</p

Measured somatic hypermutation levels from UCA or MRCA reach substitution saturation during antibody development.

<p>To demonstrate the dynamic changes of somatic hypermutation level (SHM) over time, the mean and 95% confidence interval of SHM was calculated for each time point of the three lineages. Stages from the same chain of a lineage were connected to reveal longitudinal trends. The plots of CDR regions were colored magenta while plots of FWRs were colored blue. (A) Longitudinal somatic hypermutation levels of VRC26 heavy (left) and light (right) chains. The measured SHM against UCA reached substitution saturation around 159 wpi. (B) Longitudinal somatic hypermutation levels of CH103 heavy (left) and light (right) chains. The SHM level from light chain UCA reached substitution saturation around 92 wpi, but the SHM level from heavy chain UCA appears to not reach substitution saturation. (C) Longitudinal somatic hypermutation levels of VRC01 clade 03+06 heavy (left) and light (right) chains. (D) Longitudinal somatic hypermutation levels of VRC01 clade 08 heavy (left) and light (right) chains. (E) Longitudinal somatic hypermutation levels of VRC01 clade H3. (F) Longitudinal somatic hypermutation levels of VRC01 clade L3. The measured SHM from the MRCAs of each clade reached substitution saturation around 2006.</p

The evolutionary rates of CDRs and FWRs of the three lineages decrease over time.

<p>The mean and 95% highest probability density interval (HPD) are shown for each stage. Stages from the same chain of a lineage are connected to reveal longitudinal trend. The plots of CDR regions are colored magenta while plots of FWRs are colored blue. (A) The mean evolutionary rates for the CDRs and FWRs of the VRC26 heavy (left) and light (right) chains showed about 80% decrease over the study period. (B) The mean evolutionary rate dynamics for the CDRs and FWRs of the CH103 heavy (left) and light (right) chains showed about 45% decrease over the study period. (C) Evolutionary rate dynamics for the CDRs and FWRs of the VRC01 clade 03+06 heavy (left) and light (right) chains showed about 45% decrease over the study period. But the evolutionary rate decrease for light chain CDRs is not significant. (D) Evolutionary rate dynamics for the CDRs and FWRs of the clade 08 heavy (left) and light (right) chains. The decreases in evolutionary rates are only significant for both regions in heavy chain. (E) and (F) show evolutionary rate dynamics for the CDRs and FWRs of the VRC01 clade H3 and L3, respectively. No significant decrease in evolutionary rate was detected for the two clades.</p

Study periods of the VRC26, CH103, and VRC01 lineages.

<p>To study the dynamics of evolutionary rate, selection pressure and mutability over time, we separated the time points of a lineage into two or three stages. (A) Time points of the CAP256-VRC26 lineage for which curated antibody variable region sequences were available. The eight time points were divided into three stages, which are listed above the time scale. (B) Time points of the CH103 lineage, with sequence data available at three time points, which were divided to two stages. (C). Study period of the VRC01 lineage. Since the exact date of HIV-1 infection is unavailable for this donor, the infection period before VRC01 lineage study initiation was represented with a dashed line. The approximate lineage initiation times for the VRC26 and CH103 lineages were labeled with red boxes.</p