Review Reports - Characterization of Anti-Phospholipid Antibodies in Lyme Borreliosis Using In-House Developed ELISAs

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The study of describes the development and optimization of in-house ELISAs for the detection of antiphospholipid antibodies (aPLs) directed against cardiolipin (CL), phosphatidic acid (PA), phosphatidylcholine (PC), and phosphatidylserine (PS) in patients with Lyme borreliosis. The study further evaluates differences in aPL profiles across distinct clinical manifestations of Lyme disease, including erythema migrans (EM), multiple erythema migrans (MEM), and acrodermatitis chronica atrophicans (ACA).

The manuscript may benefit from inclusion of recent studies investigating the pathogenic signaling properties of antiphospholipid antibodies in infection-associated thromboinflammatory conditions.

In particular, evidence demonstrating aPL-mediated platelet activation and MAPK signaling in infection-related vascular complications (Capozzi et al., Front Immunol, 2023) may provide additional mechanistic context for the biological relevance of the antibodies detected in Lyme borreliosis patients.

Author Response

Comments:

The manuscript may benefit from inclusion of recent studies investigating the pathogenic signaling properties of antiphospholipid antibodies in infection-associated thromboinflammatory conditions.

Response:

Dear Reviewer,

Thank you very much for your thoughtful and constructive comment. We appreciate your suggestion to include recent studies investigating the pathogenic signaling properties of antiphospholipid antibodies, including the work by Capozzi et al. (Front Immunol, 2023), which provides important insights into MAPK‑mediated platelet activation in COVID‑19–associated thromboinflammation.

We fully agree that mechanistic studies of aPL‑mediated cellular activation are highly relevant in the broader context of infection‑associated autoimmunity. However, our study was designed to:

develop, standardize, and analytically validate in‑house ELISAs for aCL, aPA, aPC, and aPS,
characterize the serological profiles of aPL across clinical stages of Lyme borreliosis,
and evaluate their potential diagnostic applicability, rather than investigate their pathogenic mechanisms.

Importantly, we did not study platelet activation, intracellular signaling pathways, or thromboinflammatory complications in Lyme borreliosis. Moreover, the biological context differs substantially: Capozzi et al. examined viral infection–associated thrombosis (COVID‑19), whereas Lyme borreliosis represents a bacterial infection with a distinct immunopathogenesis, where aPLs appear to arise primarily due to lipid scavenging by Borrelia, not due to systemic thromboinflammatory activation.

For these reasons, integrating mechanistic data on MAPK‑mediated platelet activation from COVID‑19 into our manuscript would not directly support or contextualize our diagnostic findings and might risk implying pathogenic mechanisms that we did not investigate.

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript investigates antiphospholipid antibody responses in Lyme borreliosis using newly developed in-house ELISAs targeting cardiolipin, phosphatidic acid, phosphatidylcholine, and phosphatidylserine. The topic is interesting, particularly given the growing interest in host lipid interactions in Borrelia infection. The manuscript is well organized, and the study includes several strengths, including evaluation of different clinical stages of Lyme borreliosis, assessment of both IgG and IgM isotypes, and ROC curve analysis. This is an interesting and potentially valuable study that expands knowledge regarding antiphospholipid antibodies in Lyme borreliosis. However, some minor issues should be addressed before the manuscript can be considered for publication.

In the abstract and methods, 40 healthy donors are mentioned, whereas Table 1 lists HBD, n=10. This should be corrected for clarity.
The biological interpretation of persistent or elevated IgM antiphospholipid antibodies in late Lyme borreliosis requires further discussion. Persistent IgM responses are somewhat unexpected in chronic disease and may reflect ongoing immune activation, assay cross-reactivity, or nonspecific polyclonal activation.
Figure legends should include more statistical details, including exact statistical tests, the meaning of error bars,
Supplementary Table S1 should be summarized more clearly in the main text.

Author Response

Comments

1. In the abstract and methods, 40 healthy donors are mentioned, whereas Table 1 lists HBD, n=10. This should be corrected for clarity.

Response: Thank you for your careful observation. We have corrected the mistake in Table 1. The correct No. is 40.

2. The biological interpretation of persistent or elevated IgM antiphospholipid antibodies in late Lyme borreliosis requires further discussion. Persistent IgM responses are somewhat unexpected in chronic disease and may reflect ongoing immune activation, assay cross-reactivity, or nonspecific polyclonal activation.

Response: Thank you for this important comment. We agree that the presence of IgM antiphospholipid antibodies in late Lyme borreliosis warrants clarification. We have now expanded the Discussion to address this point. Persistent or elevated IgM responses in Lyme borreliosis have been described previously and may reflect several mechanisms relevant to Borrelia infection, including:

ongoing immune stimulation due to the chronic inflammatory environment characteristic of ACA,
polyclonal B‑cell activation, a well‑recognized feature of Lyme borreliosis,
cross‑reactivity with host‑derived phospholipids, which Borrelia incorporates into its membrane,
or non‑classical IgM persistence, which has been reported in other infection‑associated autoantibody responses.

Importantly, our study was designed to evaluate the diagnostic performance of antiphospholipid antibodies rather than to dissect their immunological origin. We have therefore added a concise paragraph acknowledging these possible explanations.

‘’Although persistent IgM responses are not typically expected in late-stage disease, several mechanisms described in Borrelia infection may account for this finding. Chronic manifestations such as ACA are characterized by persistent local inflammation and ongoing antigenic stimulation, which can maintain IgM production. In addition, Borrelia burgdorferi is known to induce polyclonal B‑cell activation and to expose host‑derived phospholipids through its lipid‑scavenging biology, both of which may contribute to sustained IgM reactivity. Taken together, the inclusion of IgM provides additional insight into the temporal dynamics of the antiphospholipid antibody response in Lyme borreliosis, which was not addressed in the initial study.''

3. Figure legends should include more statistical details, including exact statistical tests, the meaning of error bars,

Response: Thank you for your comment. We have included statistical details in the figure legends and explained the graph settings.

“Group comparisons were performed using one-way ANOVA followed by Tukey’s multiple comparison test. The horizontal lines represent median values.”

4. Supplementary Table S1 should be summarized more clearly in the main text.

Response: Thank you for this comment. We agree that clarity in reporting assay precision is important. In the revised version, we ensured that the key findings from Supplementary Table S1 are explicitly summarized in the main text.