Glycosylated IgG antibodies accelerated the recovery of haemorrhagic fever with renal syndrome patients
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eLife Assessment
The authors investigated the potential role of IgG N-glycosylation in Haemorrhagic Fever with Renal Syndrome (HFRS), which may offer significant insights for understanding molecular mechanisms and for the development of therapeutic strategies for this infectious disease. The findings are useful to the field, although the strength of evidence to support the findings is incomplete. Several issues need to be addressed, including more detail on the background, methods, and results. Additional statistical tests should be performed, and the conclusions should reflect the correlational findings of the paper.
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Abstract
Abstract
Haemorrhagic fever with renal syndrome (HFRS) is a fatal disease caused by Hantaan virus (HTNV) infection. Humoral immunity is essential for effective viral clearance, but the glycosylation characteristics of immunoglobulin G (IgG) in HFRS patients is not well known. Peripheral blood mononuclear cells from HFRS patients were obtained for B subset analysis using scRNA-seq and flow cytometry. HTNV specific IgG antibody titers were detected by enzyme linked immunosorbent assay, and IgG glycosylation was analyzed by ultra-performance liquid chromatography. The proportions of the antibody-secreting memory (ASM) B cells and plasmablasts (PB) were significantly expanded among acute HFRS patients. We discovered significantly increased fucosylated IgG and decreased bisecting N-acetylglucosamine during the convalescent phase of HTNV infection. However, positive correlations were observed between ASM subsets and galactosylation/sialylation in the IgG Fc region, and between PB subsets and sialylation. Notably, the glycosylation-related genes were primarily differentially expressed in the ASM and PB subclusters, such as PRN1 and PRN2, which were enriched in the N-glycosylation modifications of proteins through asparagine. Our findings indicated that IgG N-glycosylation may play a crucial role in combating HTNV infection and contributing to clinical recovery, which provided new insights for optimizing glycoengineered therapeutic antibodies.
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eLife Assessment
The authors investigated the potential role of IgG N-glycosylation in Haemorrhagic Fever with Renal Syndrome (HFRS), which may offer significant insights for understanding molecular mechanisms and for the development of therapeutic strategies for this infectious disease. The findings are useful to the field, although the strength of evidence to support the findings is incomplete. Several issues need to be addressed, including more detail on the background, methods, and results. Additional statistical tests should be performed, and the conclusions should reflect the correlational findings of the paper.
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Reviewer #1 (Public review):
Summary:
The authors investigated the potential role of IgG N-glycosylation in Haemorrhagic Fever with Renal Syndrome (HFRS), which may offer significant insights for understanding molecular mechanisms and for the development of therapeutic strategies for this infectious disease. However, several issues need to be addressed.
Major Points:
(1) The authors should provide a detailed description of the pathogenesis of Haemorrhagic Fever with Renal Syndrome (HFRS) and elaborate on the crucial role of IgG proteins in the disease's progression (line 65).
(2) An additional discussion on the significance of glycosylation, particularly IgG N-glycosylation, in viral infections should be included in the Introduction section.
(3) In the Abstract section, the authors state that HTNV-specific IgG antibody titers were …
Reviewer #1 (Public review):
Summary:
The authors investigated the potential role of IgG N-glycosylation in Haemorrhagic Fever with Renal Syndrome (HFRS), which may offer significant insights for understanding molecular mechanisms and for the development of therapeutic strategies for this infectious disease. However, several issues need to be addressed.
Major Points:
(1) The authors should provide a detailed description of the pathogenesis of Haemorrhagic Fever with Renal Syndrome (HFRS) and elaborate on the crucial role of IgG proteins in the disease's progression (line 65).
(2) An additional discussion on the significance of glycosylation, particularly IgG N-glycosylation, in viral infections should be included in the Introduction section.
(3) In the Abstract section, the authors state that HTNV-specific IgG antibody titers were detected and IgG N-glycosylation was analyzed. However, the analysis of plasma IgG N-glycans is described in the Methods section. Therefore, the authors should clarify the glycome analysis process. Was the specific IgG glycome profile similar to the total IgG N-glycome? Given the biological relevance of specific IgG in immunological diseases, characterizing the specific IgG N-glycome profile would be more significant than analyzing the total plasma IgG.
(4) Further details regarding the N-glycome analysis should be provided, including the quantity of IgG protein used and the methodology employed for analyzing IgG N-glycans (lines 286-287).
(5) Additional statistical analyses should be performed, including multiple comparisons with p-value adjustment, false discovery rate (FDR) control, and Pearson correlation (line 291).
(6) Quality control should be conducted prior to the IgG N-glycome analysis. Additionally, both biological and technical replicates are essential to assess the reproducibility and robustness of the methods.
(7) Multiple regression analysis should be conducted to evaluate the influence of genetic and environmental factors on the IgG N-glycome.
(8) Line 196. Additional discussions should be included, focusing on the underlying correlation between the differential expression of B-cell glycogenes and the dysregulated IgG N-glycome profile, as well as the potential molecular mechanisms of IgG N-glycosylation in the development of HFRS.
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Reviewer #2 (Public review):
Summary:
This work sought to explore antibody responses in the context of hemorrhagic fever with renal syndrome (HFRS) - a severe disease caused by Hantaan virus infection. Little is known about the characteristics or functional relevance of IgG Fc glycosylation in HFRS. To address this gap, the authors analyzed samples from 65 patients with HFRS spanning the acute and convalescent phases of disease via IgG Fc glycan analysis, scRNAseq, and flow cytometry. The authors observed changes in Fc glycosylation (increased fucosylation and decreased bisection) coinciding with a 4-fold or greater increase in Haantan virus-specific antibody titer. They suggest that these shifts contribute to disease recovery. The study also includes exploratory analyses linking IgG glycan profiles to glycosylation-related gene …
Reviewer #2 (Public review):
Summary:
This work sought to explore antibody responses in the context of hemorrhagic fever with renal syndrome (HFRS) - a severe disease caused by Hantaan virus infection. Little is known about the characteristics or functional relevance of IgG Fc glycosylation in HFRS. To address this gap, the authors analyzed samples from 65 patients with HFRS spanning the acute and convalescent phases of disease via IgG Fc glycan analysis, scRNAseq, and flow cytometry. The authors observed changes in Fc glycosylation (increased fucosylation and decreased bisection) coinciding with a 4-fold or greater increase in Haantan virus-specific antibody titer. They suggest that these shifts contribute to disease recovery. The study also includes exploratory analyses linking IgG glycan profiles to glycosylation-related gene expression in distinct B cell subsets, using single-cell transcriptomics. Overall, this is an interesting study that combines serological profiling with transcriptomic data to shed light on humoral immune responses in an underexplored infectious disease. The integration of Fc glycosylation data with single-cell transcriptomic data is a strength. However, some improvements could be made in the clarity of both the Results and Materials and Methods sections, and some conclusions would benefit from greater caution, particularly in avoiding overinterpretation of correlative findings.
Comments:
(1) While it is great to reference prior publications in the Materials and Methods section, the current level of detail is insufficient to clearly understand the study design and experimental procedures performed. Readers should not be expected to consult multiple previous papers to grasp the core methodological aspects of the present paper. For instance, the categorization of HFRS patients into different clinical subtypes/courses, and the methods for measuring Fc glycosylation should be explicitly described in the Materials and Methods section of this manuscript.
(2) The authors should explain the nature of their cohort in a bit more detail. While it appears that HFRS cases were identified based on IgM ELISA and/or PCR, these are indicators of the Haantan virus infection. My understanding is that not all Haantan virus infections progress to HFRS. Thus, it is unclear whether all patients in the HFRS group actually had hemorrhagic fever. This distinction is critical for interpreting how the results observed relate to disease severity.
(3) The authors state that: "A 4-fold or greater increase in HTNV-NP-specific antibody titers usually indicates a protective humoral immune response during the acute phase", but they do not cite any references or provide any context that supports this claim. Given that in their own words, one of the most significant findings in the study is changes in glycosylation coinciding with this 4-fold increase, it is important to ground this claim in evidence. Without this, the use of a 4-fold threshold appears arbitrary and weakens the rationale for using this immune state as a proxy for protective immunity.
(4) The authors also claim that changes in Fc glycosylation influence recovery from HFRS - a point even emphasized in the manuscript title. However, this conclusion is not well supported by the data for two main reasons. First, the authors appear to measure bulk IgG Fc glycans, not Fc glycans of Hantaan virus-specific antibodies. While reasonable, this is something that should be communicated in the manuscript. Hantaan virus-specific antibodies are likely a very small fraction of total circulating IgG antibodies (perhaps ~1%), even during acute infection. As a result, changes in bulk Fc glycosylation may (or may not) accurately reflect the glycosylation state of Hantaan virus-specific antibodies. Second, even if the bulk Fc glycan shifts do mirror those of Hantaan virus-specific antibodies, it remains unclear whether these changes causally drive recovery or are merely a consequence of the infection being resolved. Thus, while the differences in Fc glycosylation observed are interesting - and it is tempting to speculate on their functional significance - the manuscript treats the observed correlations as causal mechanistic insight without sufficient data or justification.
(5) Fc glycosylation is known to be influenced by covariates such as age and sex. While it is helpful that the authors stratified the patients by age group and looked for significant differences in glycosylation across them, a more robust approach would be to directly control for these covariates in the statistical analysis - such as by using a linear mixed effects model, in which disease state (e.g., acute vs. convalescent), age, and sex are treated as fixed effects, and subject ID is included as a random effect to account for repeated measures. This would allow the authors to assess whether observed differences in Fc glycosylation remain significant after accounting for potential confounders. This could be important given that some of the reported differences are quite small, for example, 94.29% vs. 94.89% fucosylation.
(6) The manuscript states that there are limited studies on antibody glycosylation in the context of HFRS, but does not cite any relevant literature. If prior work exists, it should be cited to contextualize the current study. If no prior studies have been conducted/reported, to the author's knowledge, that should be stated explicitly to show the novelty of the work.
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