Germline-somatic residue synergy reshapes antibody encounter-state pathways to enhance HIV-1 recognition

Reviewer #1 (Public review):

Summary:

This manuscript uses simulations and MSMs paired with experimental binding assays to examine the binding mechanisms of different antibodies to their targets. The authors argue that contacts in encounter complexes play an important role in determining the association rates and binding affinities that distinguish more mature antibodies from less efficacious antibodies from earlier in the maturation process.

Strengths:

The idea is interesting, and the combination of computational models and experiments is a good direction.

Weaknesses:

The manuscript focuses heavily on kinetics, but it is not clear whether the simulations recapitulate the relative rates of binding of the two antibodies. The relationship between the simulated binding behavior and the experimentally observed kinetic differences is therefore not fully established.

The comparison of committor probabilities or fluxes between the two antibodies may not be appropriate. These properties are related to the barrier height the system has to cross to move forward vs back to the starting state, under the simplifying assumption that the properties of other states aren't critical. Even in this simplified case, the same flux or committor probability could occur with very different barrier heights, e.g., rates or transition probabilities.

Some claims are presented in a very qualitative way that people who aren't experts in MSMs may have difficulty tying to the results in Figure 1.

Reviewer #2 (Public review):

Summary:

The manuscript addresses an important and underexplored question: how affinity maturation alters antibody encounter-state landscapes rather than simply improving bound-state affinity. The authors combine adaptive MD, Markov State Models (MSMs), transition path theory, mutagenesis, SPR kinetics, and double-mutant cycle analysis into a coherent story.

Strengths:

This manuscript presents a compelling computational and experimental analysis of antibody affinity maturation in the HIV-1 DH270 lineage. The main finding is that somatic mutations reshape encounter-state pathways through glycan-mediated steering rather than simply stabilizing the final bound state. This is novel and potentially important for vaccine design. The combination of adaptive MD, MSMs, SPR kinetics, and double-mutant cycle analysis is a major strength.

Weaknesses:

The proposed sequence that somatic mutations cause glycan capture, which causes reorientation, which causes enhanced association, is based on correlation rather than direct causality.

The four MSM states are not convincingly explained, and the robustness of these states is unclear.

The productive collision surface area analysis needs more quantitative data.

The coupling energy values are near the uncertainty range. Some conclusions about long-range communication networks appear stronger than the data justify. The data support coupling, but they do not necessarily support detailed mechanistic networks.

The study investigates one lineage, one epitope class, and one viral system. Hence, the generalization is limited.

Reviewer #3 (Public review):

Summary:

In this work, the authors set out to characterise how encounter states between antibodies and antigens evolve during affinity maturation through molecular dynamics simulations and Markov state modeling. They demonstrate how early glycan-mediated interactions increased association rates rather than modifying the final bound state.

Strengths:

The computational approach is backed up by experimental results and allows for visualising otherwise too short-lived association states, thus allowing to discriminate between different lineages.

Weaknesses:

The figures and captions are not always clear about what they are trying to show. The choice of CVs is not sufficiently discussed.