RR:C19 Evidence Scale rating by reviewer:
Reliable. The main study claims are generally justified by its methods and data. The results and conclusions are likely to be similar to the hypothetical ideal study. There are some minor caveats or limitations, but they would/do not change the major claims of the study. The study provides sufficient strength of evidence on its own that its main claims should be considered actionable, with some room for future revision.
Review: Development of neutralization breadth against HIV is a difficult and poorly understood challenge for the field. This paper presents evidence that MT145K and MT145K.dV5 Envs were better at eliciting and expanding B cells with favorable bnAb properties than MT145 in the setting of SCIV infection in rhesus macaques. However, in the end, plasma neutralization breadth did not develop.
This paper describes the characterization of B cells in rhesus macaques that were infected with one of three SIVmac-SIVcpz Env chimeras (SCIVs) expressing Envs MT145, MT145K, and MT145K.dV5. The corresponding MT145K Env SOSIP trimer immunogen has been shown to engage V2 bnAb precursors in knock in mouse models. The MT145K.dV5 is a next generation version that knocks out an immunodominant epitope in V5. In the ongoing effort to develop strategies to elicit V2 apex bnAbs, the authors aimed to test whether the MT145K and MT145K.dV5 SCIVs would produce more V2 bnAb like precursors than MT145 in the setting of infection with ongoing viral diversity, with the ultimate goal being plasma neutralization breadth and a bnAb developing over time. Here, they examined clonal expansion of B cells with long CDRH3 regions, and other potentially favorable properties such as use of certain D regions and tyrosine sulfation motifs. To this end, the paper presents a large amount of data and broad analyses spanning longitudinal Env variation, RepSeq of bulk IgG and IgM B cells, 10X Genomics VDJ sequencing of antigen specific B cells and single antigen specific B cell sorts into culture for IgG secretion and recovery of mAbs for the MT145K.dV5 SCIV infected RMs. The paper is technically sound, presents intriguing findings, and represents a novel approach to developing and evaluating HIV immunogens.
The paper presents some evidence that MT145K and MT145K.dV5 SCIVs were better at eliciting and expanding B cells with long CDRH3 and other properties than MT145. A closer look at MT145K.dV5 SCIV infected animals reflected this propensity in an analysis of antigen specific B cells, some of which shared features with HIV V2 bnAbs. However, in the end, plasma neutralization breadth was not produced during the infection. This study demonstrates the use of SCIVs to study B cell dynamics and supports that these immunogens could have utility in strategies to elicit neutralization breadth in this outbred model that closely mimics human B cell and antibody responses. This study also highlights the difficulties in eliciting bnAbs and underscores our limited understanding of how to recapitulate this process. Major and minor comments listed below are areas that could be considered to strengthen the conclusions of the manuscript as well as polish up the text.
It would be interesting to show the neutralization data for the recovered mAbs. This reviewer could not find supplementary table 1, which is referenced for this data. The data seems to only be shown indirectly in an upset plot that does not present potency/IC50. This data could be added to Fig. 6A. Also, was the CRF250 Env pseudovirus neutralized by some mAbs the wildtype version or the N160K mutant as used in Supplementary Fig. 1?
The MT145K.dV5 infected animals developed relatively low autologous plasma neutralization titers. Which of these RMs were used for antigen specific B cell sorting and recovery of mAbs/B cell cultures? Why are the plasma ID50s for these 3 RMs not shown prior to week 24 in Suppl. Fig. 1? Also, could the authors comment on why the preference for expansion of longer CDRH3s with other favorable properties did not lead to more robust autologous neutralization in these animals?
Could the authors comment on the significance of the examination of the bulk repertoire by RepSeq, as it is not known whether these B cells were responding to Env, and present more clearly how well the IgG repertoire sequencing matched up with antigen specific B cells? Were singlets included in this analysis? If RepSeq lineages did not show up as antigen specific in situations where those assays were performed, were they still considered in the analysis?
Importantly, it was not clearly stated where the VDJ data sets would be deposited for public access. The RepSeq and 10X Genomics VDJ data sets need to be deposited into appropriate public databases such as SRA and GEO.
Were there differences in plasma viral load or CD4 T cell depletion between groups that could have impacted the skewing of B cell responses towards or away from long CDRH3 and the development of neutralization?
How do the rhesus macaque long CDRH3 antibodies elicited by MT145K or MT145K.d5 Envs compare directly with the human V2 bnAbs in terms of VH family, D and J regions, identity, other features? Especially the mAbs known to have or lack neutralizing activity, dependence on N160, etc. Maybe could present this in an amino acid alignment for direct comparison?
There was no mention of the light chain usage and whether that was consistent or notable in any way.
What is the scale of the phylogenetic trees and are they showing distance from germline, MRCA, etc.? Do the color scales and trees depict the same parameter?
Should be consistent throughout the manuscript about abbreviating rhesus macaque/RM.
Should be consistent about SGA and SGS.
Line 276 – ‘Overall’ should be capitalized.
Line 993 – Expi293Fs should be in 8% CO2?
Line 996 – supernatant ‘was passed through’?
Figure 1 legend – Citation needs to be formatted.
Line 94 to 95 – regarding native-like trimers that elicit autologous neutralizing antibody responses – should specify whether you are referring to studies in rhesus macaques and check whether the cited references are most appropriate.
Line 117 – Should be SIVcpz?
Line 931 – Should be TZM-bl
Line 400 – Should this be Fig 5G-I?
Line 403 – Remove hyphen between ‘asked what’
Line 457 – Should be interclonal?
In methods, check units, particularly the neutralizing antibody assay methods paragraph.
Line 979 – Should be SIVcpz Env SOSIP trimers? Co-transfected with furin expression plasmid?
Line 991 – Need to define HC and LC.
Line 1079 –Citation needs to be formatted.
Line 1075 – What version of CellRanger was used to analyze the VDJ libraries and were any custom steps used?