SARS-CoV-2 and HCoV IgG Antibodies in the Breast Milk of a Postpartum SARS-CoV-2 Patient Following Bamlanivimab Administration: A Case Report
, Christopher Condin 2,3, Ana Citlali Márquez 4, Susan Li 2,3, Nimrat Binning 2,3, Miriam Gibson 2,3, Brayden Griffiths 2,3, Alissa Wright 5, Deborah Money 6,7, Mel Krajden 1,4, Muhammad Morshed 1,4, Agatha N. Jassem 1,4, Gregory Haljan 2,3,5 and Inna Sekirov 1,4,*
Round 1
Reviewer 1 Report
Comments and Suggestions for Authors1) The introduction lacks adequate referencing. Numerous studies have examined the composition of breast milk during the SARS-CoV-2 pandemic—not only in relation to the immune response, but also focusing on other antimicrobial proteins and cytokines present in milk following vaccination or infection. The background section should be strengthened with more comprehensive and up-to-date literature to better support the context.
2) Line 83: The milk collection process needs to be described in greater detail. Information such as the volume collected, whether the sample was taken from the midstream or the foremilk (which is more water-rich), whether it was self-collected, and the specific conditions under which it was obtained should be clearly specified. The current description lacks sufficient data, and multiple variables could significantly affect the milk's composition.
3) What were the storage conditions for the milk samples?
4) Were the milk samples processed prior to antibody measurement? If so, what processing steps were taken? Both the sample preparation and the antibody quantification methods should be described in detail.
5) By day 3 following symptom onset, it is generally too early for most individuals to exhibit significant IgG seroconversion. Typically, IgM antibodies emerge around days 5–7 post-infection, while IgG antibodies develop between days 7–14. Therefore, the absence of IgG in breast milk on day 3 is consistent with the expected immune response timeline.If IgG is then detected on day 7 post-symptom onset—immediately after administration of bamlanivimab—it becomes more challenging to distinguish whether the antibodies are the result of early seroconversion or passive transfer of the monoclonal antibody. Without an assay that specifically differentiates between endogenous IgG and exogenously administered bamlanivimab, we cannot conclusively determine whether the IgG detected in the milk reflects the mother's own immune response or the presence of the therapeutic antibody.
6) I would appreciate it if Figures 1b–1d could be enlarged, as they are currently difficult to see and lack clarity.
7) When was the case recorded/during what period was the study conducted?
Author Response
We greatly appreciate the reviewer for taking the time to assess our manuscript and providing insightful feedback and suggestions. We have carefully addressed all the reviewer comments, and our responses are stated in blue below. Please note that all the line and page numbers we mention in this reply refer to our revised manuscript and not the original paper, unless specified. Additionally, we realized that references did not display correctly in the original submission due to syncing issues, we sincerely apologize for this oversight and have carefully reviewed and corrected the in-text citations to ensure that the reference list now accurately reflects the cited studies.
1) The introduction lacks adequate referencing. Numerous studies have examined the composition of breast milk during the SARS-CoV-2 pandemic—not only in relation to the immune response, but also focusing on other antimicrobial proteins and cytokines present in milk following vaccination or infection. The background section should be strengthened with more comprehensive and up-to-date literature to better support the context.
Response 1: We thank the reviewer for this helpful suggestion. In response, we have added citations to several recent studies characterizing a wide range of immune-related factors in breast milk following SARS-CoV-2 infection or vaccination. While we recognize the broader relevance of antimicrobial proteins and cytokines in breast milk, we have opted to briefly acknowledge these findings without going into extensive detail to maintain the focus on antibody responses, which is the primary objective and scope of our study. Nonetheless, we thank the reviewer’s suggestions as these additions indeed strengthen the background, while maintaining its alignment with our study’s scope.
Line 48-49: along with a variety of immune cells and cytokines [7,8], highlighting the complex immunological profiles within human milk following exposure to SARS-CoV-2.
Citations added:
- Graciliano NG, Goulart MOF, de Oliveira ACM. Impact of Maternal Exposure to SARS-CoV-2 on Immunological Components of Breast Milk. Int J Mol Sci. 2025 Mar 13;26(6):2600.
- Armistead B, Jiang Y, Carlson M, Ford ES, Jani S, Houck J, et al. Spike-specific T cells are enriched in breastmilk following SARS-CoV-2 mRNA vaccination. Mucosal Immunol. 2023 Feb;16(1):39–49.
2) Line 83: The milk collection process needs to be described in greater detail. Information such as the volume collected, whether the sample was taken from the midstream or the foremilk (which is more water-rich), whether it was self-collected, and the specific conditions under which it was obtained should be clearly specified. The current description lacks sufficient data, and multiple variables could significantly affect the milk's composition.
Response 2: Thank you for the comment. We have addressed this and have provided more methodological details as follows:
Lines 89-90: “All breast milk samples were self-collected and kept at -20°C until processing and testing. No specific instructions for self-collection were provided to the patient, and approximately 50mL was collected at each time point”
As this was a relatively large volume, all the samples likely included a mixture of foremilk, midstream milk, and hindmilk, rather than a targeted collection of midstream or foremilk specifically. As such, the composition likely stayed fairly consistent throughout the samples. Additionally, breast milk collection started 9 days after delivery, as such, this is unlikely to be colostrum.
3) What were the storage conditions for the milk samples?
Response 3: Thank you to the reviewer for this question. The milk samples were stored at -20C until testing, which was performed shortly after all samples had been conducted. This has been consistent with standard practice in other breast milk antibody research, and previous studies have demonstrated stable immunological properties in breast milk for at least 6 months.
We have included this in lines 90-96: “All breast milk samples were self-collected by the mother and kept at -20°C until testing. Testing was carried out in the fall of 2021, shortly after the collection of all samples.”
4) Were the milk samples processed prior to antibody measurement? If so, what processing steps were taken? Both the sample preparation and the antibody quantification methods should be described in detail.
Response 4: Thank you to the reviewer for pointing out this gap in our manuscript, we would like to apologize for the lack of clarity. Milk samples were processed prior to antibody measurement using validated methods (validation manuscript in progress to be submitted by the end of July 2025), which have also been used in other research studies we’ve collaborated on (Korchinski et al, 2024, Vaccine).
We have improved our methods section accordingly to better describe both the milk sample preparation and antibody quantification in detail, as follows:
Lines 90-118: “All breast milk samples were self-collected by the mother and kept at -20°C until testing. No specific instructions for self-collection were provided to the patient, and approximately 50mL was provided at each timepoint. Testing was carried out following the collection of all samples in the fall of 2021, shortly after the collection of all samples. Breast milk samples were thawed at 37°C, centrifuged at 1300 RCF for 20 minutes to separate the opaque cream (top) and aqueous (bottom) layers. The top layer was subsequently discarded, and the remaining aqueous layer was used for antibody testing. Processing methods yielded approximately up to 50% of IgG recovery, which had been demonstrated in validation studies testing breast milk spiked with known amounts of IgG.
Processed breast milk and serum IgG antibodies against the spike, RBD, and nucleocapsid regions of SARS-CoV-2, along with the spike protein of four HCoVs (HCoV-229E, HCoV-NL63, HCoV-HKU1, and HCoV-OC43), were quantified using the Meso Scale Discovery (MSD) V-PLEX Coronavirus Panel 2 (Rockville, USA), an electrochemiluminescent multiplex immunoassay that has been previously validated and demonstrated for research use on milk and serum samples. Briefly, antigens were printed on the bottom of a multi-spot 96 well plate from MSD, and the assay was performed according to manufacturer’s instructions. Following plate blocking, processed milk (diluted 1:100) and serum (diluted at 1:5000) specimens were added and incubated for two hours. Bound antibodies were then labelled with SULFO-TAG™ Anti-human IgG for an hour. MSD Gold Read buffer was added after a final wash and plates were immediately read using the MSD QuickPlex SQ120. All incubation steps were carried out shaking at room temperature (20-26°C) and all washes were performed using the MSD Wash Buffer. Raw data was imported onto the MSD Discovery Workbench software for data processing. Figures were prepared on GraphPad Prism 10.”
5) By day 3 following symptom onset, it is generally too early for most individuals to exhibit significant IgG seroconversion. Typically, IgM antibodies emerge around days 5–7 post-infection, while IgG antibodies develop between days 7–14. Therefore, the absence of IgG in breast milk on day 3 is consistent with the expected immune response timeline. If IgG is then detected on day 7 post-symptom onset—immediately after administration of bamlanivimab—it becomes more challenging to distinguish whether the antibodies are the result of early seroconversion or passive transfer of the monoclonal antibody. Without an assay that specifically differentiates between endogenous IgG and exogenously administered bamlanivimab, we cannot conclusively determine whether the IgG detected in the milk reflects the mother's own immune response or the presence of the therapeutic antibody.
Response 5: We thank the reviewer for this insightful comment. The authors agree with the reviewer’s comments and have incorporated this within our discussion as a limitation of our work in lines 188-197:
This is consistent with the expected timeline for SARS-CoV-2 IgG response during a primary immune response, which typically arises around days 7-14 post-infection, reaching peak levels between 21 to 49 days post symptom onset [23,25]. While our methods do not conclusively differentiate between endogenous IgG from the mother’s natural immune response or exogenously administered bamlanivinab, we observed a sharp decline following the initial spike in antibodies after day 3 likely represented by declining bamlanivimab. As such, antibodies following this likely represents both en-dogenous and exogenous antibodies, though the relative level of bamlanivimab is likely much lower compared to that of the natural antibody response by the second peak.
6) I would appreciate it if Figures 1b–1d could be enlarged, as they are currently difficult to see and lack clarity.
Response 6: The authors agree and thank the reviewer for this suggestion – the figures have been modified accordingly.
7) When was the case recorded/during what period was the study conducted?
Response 7: This case was recorded in May 2021, and the study was conducted shortly after all samples were collected, in the fall of 2021.
We have included this in line 80 of our manuscript: “In May 2021, she tested positive by PCR for SARS-CoV-2...” and in lines 92-93 “Testing was carried out following the collection of all samples in the fall of 2021.”
Reviewer 2 Report
Comments and Suggestions for AuthorsThank you for the opportunity to review this case report. I commend the authors for presenting a timely and methodologically sound case report on the longitudinal detection of SARS-CoV-2 and HCoV antibodies in breast milk following the administration of monoclonal antibodies. The report addresses a unique niche in maternal-infant immunity during the COVID-19 pandemic and contributes novel data to the field. I have minor queries to improve this paper
- Consider adding standard error band for the graph
- Enhance the clarity of the graph by making it in one dedicated page with landscape layout.
Author Response
We greatly appreciate the reviewer for taking the time to assess our manuscript and providing insightful feedback. We have carefully addressed all the reviewer comments, and our responses are stated in blue below. Please note that all the line and page numbers we mention in this reply refer to our revised manuscript and not the original paper, unless specified. Additionally, we realized that references did not display correctly in the original submission due to syncing issues, we sincerely apologize for this oversight and have carefully reviewed and corrected the in-text citations to ensure that the reference list now accurately reflects the cited studies.
Thank you for the opportunity to review this case report. I commend the authors for presenting a timely and methodologically sound case report on the longitudinal detection of SARS-CoV-2 and HCoV antibodies in breast milk following the administration of monoclonal antibodies. The report addresses a unique niche in maternal-infant immunity during the COVID-19 pandemic and contributes novel data to the field. I have minor queries to improve this paper
- Consider adding standard error band for the graph
Response 1: We thank the reviewer for this insightful suggestion. However, the dataset represents a single case with one sample collected and tested per time point. Additionally, as we used a validated assay, there are no replicates available to compute variability measures. As such, it is not possible to derive standard deviations, standard errors, or confidence intervals to generate error bands for these figures.
- Enhance the clarity of the graph by making it in one dedicated page with landscape layout.
Response 2: Thank you to the reviewer for this comment, the authors agree that enlarging the clarity of the graphs will help improve the paper. We have addressed this accordingly and modified the figure layout and size for clarity, but we maintained the figure in portrait layout for readability.
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsIt is my view that a more rigorous review of the bibliographic references could have been undertaken, with a focus on selecting high-impact articles — specifically those with substantial citation counts or authored by researchers specializing in the study of breast milk in the context of SARS-CoV-2 infection.
