PREreview of Increase in SARS-CoV-2 RBD-specific IgA and IgG Antibodies in Human Milk from Lactating Women Following the COVID-19 Booster Vaccination

Review of: Henle, A. 2022. “Increase in SARS-CoV-2 RBD-specific IgA and IgG Antibodies in Human Milk from Lactating Women Following the COVID-19 Booster Vaccination.” medRxiv. DOI: https://doi.org/10.1101/2022.02.23.22271414

This review was written collaboratively by undergraduates at Mount Holyoke College (MA, USA) as an assignment in a course taught by Dr. Rebeccah S. Lijek, Assistant Professor of Biological Sciences. Student-reviewers who give their permission to list their names or initials are: Nafeesah Ahmed-Adedoja, R.F, Sara Hearn, Simone Jacob, C.A.L., and Alexandria Taylor. Student-editors who compiled the independent reviews into the following response to the author and who give their permission to list their names are: Olive Aries, Madison Dresler, Rameen Farrukh, Soli Guzman, Amanda Kearney, Valentina Shrum, Siyu Yin.

COI: The review authors declare no conflict of interest and have no personal or financial relationship with the study’s author.

Summary:

This study investigates whether the Pfizer-BioNTech (BNT162b2) COVID-19 mRNA vaccine booster results in increased antibody levels in breast milk. Henle used ELISA to measure the levels of anti-receptor binding domain (RBD) specific IgG and IgA antibodies in the breast milk of 12 lactating parents before and after receiving a Pfizer-BioNTech booster vaccine. They found that both IgA and IgG levels increased over time after the booster for up to 60 days. These data support their claim that the Pfizer-BioNTech booster vaccine increases anti-RBD antibodies in human milk. This study is novel because, although other studies have tested the level of antibodies in breast milk after the initial series of Pfizer-BioNTech vaccinations, this is the first study to measure the amount of antibodies in breast milk after receiving the Pfizer-BioNTech booster. This helps demonstrate why lactating people should receive the COVID-19 vaccine booster. As of this moment, children under 5 cannot be vaccinated, so their immunity to COVID-19 could depend on antibodies acquired passively through breast milk. This study merits publication once the requested additional information about sample size limitations and the connection between antibodies found in breast milk and passive immunity in infants are included.

Strengths:

• A major strength of this manuscript is the clear writing and organization. The introduction provides an excellent overview of the issue and background, a clear statement of hypothesis, and includes where relevant past studies have left off and where the author is picking up from. The author also does a good job stating the purpose, relevance, and significance of this study.
• Results are clearly stated and straightforward to interpret. Appropriate use of statistics to show significance of data and difference between data points.
• The methods and materials section is very detailed and easily replicable based on succinct writing, to a point where researchers in the future could very likely recreate the same experiment.
• The manuscript does a good job addressing the limitations in the study & methods. 
• A strength of this study is its timely societal implication. For example, this study may help lactating parents make data-driven decisions about getting a COVID19 vaccine booster. 

Major critiques:

• Although the sample size is mentioned briefly in the limitations section it is not clear why such a small sample size was chosen. There is no mention or justification for the selection of only 12 participants, which makes the study less applicable to the wider population especially as it is not diverse. To address this weakness, the authors should at a minimum expand the section on the study size limitations. Ideally, the author could perform a power analysis on these preliminary data in order to determine the statistically appropriate sample size to detect an effect and then enroll that additional number of participants. If you are able to expand the study, it would be more impactful to do so with a more diverse population.
• It is not clear if the major conclusion is that IgA and IgG levels in human breast milk increase after getting the Pfizer booster, or the larger statement that this increase is a source of immunity for breastfeeding infants. If the former is the author's major conclusion, the data the author has here is sufficient to make that claim. However, if the author is trying to prove the latter, further experiments that show the functionality and protective capacity of the breast milk antibodies need to be conducted (e.g. neutralization assays). Either way, the manuscript requires a more robust citation of other studies that show the effect of maternal antibodies passed to the infant from breastmilk on the infant immune system since this is a major point of significance for the study.
• In the limitations, it is stated that the participants were not tested for SARS-CoV-2 by RT-PCR and that it was possible that participants could have had asymptomatic or symptomatic COVID-19. This is a major confounding variable and could compromise the results - how can the author rule out that the increase in breast milk antibodies was not due to SARS-CoV-2 infection instead of the booster vaccination? Please emphasize this as a major limitation of the study, and state that for future studies routine testing should be administered in order to ensure the absence of COVID-19 infection throughout the duration of the study. 
• There is some confusion about the relevance of the blood that was collected. It is not written clearly what is being done with the collected blood and there is no mention of it in the introduction or a graph representing the data from the blood experiment. Please add text clarifying this sample collection and any associated data analysis. 

Minor critiques:

• The second sentence under “Study design” is unclear. Were the subjects who were recruited through social media and mom groups only educators and healthcare workers? How did you ensure this through social media and mom groups? The method of selection of subjects, and reasoning for why subjects were selected in this way could be expanded on.
• At the end of the paragraph under “Milk and blood sample collection and processing”, the negative control is described as “human milk from July 2019”. This makes sense since it is pre pandemic, but for added clarity the author can specifically state that the control is milk from individuals who have not been vaccinated against COVID-19 or had COVID-19. In addition the author can clarify if the controls were stored and centrifuged in the same way as the other samples.
• The author might consider keeping the scale the same for A and B in Fig. 1 to make it clear to the reader how much more IgG is present in the milk than IgA. In addition they can change the titles of A and B to “IgA levels in human milk” and “IgG levels in human milk.”