Pre-existing antibodies and age shape the immune response following EV-A71 vaccine: a prospective serological study among the Chinese pediatric population

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1. 

   PREreview

   Nov 10, 2025

   This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/17568217.

   Summary & Methods

   This preprint illustrates the first study to model the EV-A71 vaccine, used to treat hand, foot, and mouth disease (HFMD), through the combination of pre- and post-vaccination antibody titers and age. The EV-A71 vaccine has been proven to have a high efficacy rate (over 90%), but the ways in which age and baseline antibodies affect this response is less well known. The authors used generalized additive mixed models (GAMMs) to study the link between antibody level and age and analyze vaccination responses and quantify the affect of pre-existing antibody levels in vaccine response. The study also focused on baseline titers of participants, including seropositive …

   This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/17568217.

   Summary & Methods

   This preprint illustrates the first study to model the EV-A71 vaccine, used to treat hand, foot, and mouth disease (HFMD), through the combination of pre- and post-vaccination antibody titers and age. The EV-A71 vaccine has been proven to have a high efficacy rate (over 90%), but the ways in which age and baseline antibodies affect this response is less well known. The authors used generalized additive mixed models (GAMMs) to study the link between antibody level and age and analyze vaccination responses and quantify the affect of pre-existing antibody levels in vaccine response. The study also focused on baseline titers of participants, including seropositive participants. Their research ultimately investigated the effects, if any, of pre-existing antibody titers and age at vaccination on immune responses in participants with varying pre-vaccine titer levels. 

   The study population included 1585 Chinese children aged 5-36 months from both a clinical trial and a cohort study. The participants were split into three groups based on their baseline antibody levels, including high baseline antibodies, low baseline antibodies, and undetectable baseline antibodies. Using a modified cytopathogenic effect assay, researchers measured antibody levels post 2 doses of the vaccine and measured the fold increase over pre-existing baseline titer levels. They controlled for sex, vaccination age, date of sampling, and source of data. They found that the titers post 2 doses of vaccine were highest among children in the high baseline antibody group, even though their fold increase was less than children in the low or undetectable baseline antibody group. They also found that an increase in age at vaccination, when controlling for other factors, resulted in an increase in vaccine-induced antibody titers. The researchers noted that they were only focused on short-term responses to vaccination, and that extended evidence is needed. This manuscript has long-term reference, and is innovative in terms of technical novelty and future vaccination policy making. This preprint is well written and describes a thought-out experiment to test vaccination response and administration. However, their ability to improve a few issues about design choice and literature review would greatly improve the quality of the paper. 

   Major Issues 

   1. The methods section needs more detail and references to clarify their decision to use specific modeling types of GMTs and GMFI in their methodology. I would suggest adding literature review or references to similar vaccine studies that use the same methods to defend the usage of these modeling methods to test the efficacy and understanding of the EV-A71 vaccine is the best under these modeling methods. 

      1. This reference could be sited to provide evidence of the use of titers when investigating antibody methodology: 

         1. Miyazaki H, Yamanaka G, Furukawa K, Ichiki M. Effect of vaccine program on IgG antibody titers for measles, rubella, varicella, and mumps in young adults in Japan: Survey between 2018 and 2021. J Infect Chemother. 2022 Oct;28(10):1410-1414. doi: 10.1016/j.jiac.2022.06.016. Epub 2022 Jun 29. PMID: 35779802. 

         2. Taniguchi Y, Suemori K, Tanaka K, Okamoto A, Murakami A, Miyamoto H, Takasuka Y, Yamashita M, Takenaka K. Long-term transition of antibody titers in healthcare workers following the first to fourth doses of mRNA COVID-19 vaccine: Comparison of two automated SARS-CoV-2 immunoassays. J Infect Chemother. 2023 May;29(5):534-538. doi: 10.1016/j.jiac.2023.01.007. Epub 2023 Jan 22. Erratum in: J Infect Chemother. 2024 Nov;30(11):1197. doi: 10.1016/j.jiac.2024.05.002. PMID: 36696921; PMCID: PMC9867842. 

   2. The discussion is well-written, but the authors should clarify the relationship between pre-existing antibody levels and vaccine response. For instance, the authors write that "pre-existing antibody levels may dampen the apparent antibody boosting by the EV-A71 vaccine…" I suggest that the authors address why the antibody levels may appear lower, such as a such as a ceiling effect or another sort of immunological suppression. I would recommend clarifying that this "dampening" does not necessarily imply reduced responsiveness to the vaccine, but could be a consequence of differing baseline antibody levels. If the authors were to write a few sentences about this relationship touch on this idea (whether or not they agree) in the discussion, the strength of their evidence would greatly increase. 

   Minor Issues

   1. The preprint's abstract states that "vaccine-induced responses also varied with age, showing periodic increases in children under 36 months." I would suggest that the authors explain what kind of pattern they found in vaccine-induced responses, as a "period increase" is somewhat vague. How was this pattern increase measured, for instance, and was this increase graduate, cyclical, random, etc? This would help the readers understand further how age and vaccine response are related. 

   Competing interests

   The authors declare that they have no competing interests.

   Use of Artificial Intelligence (AI)

   The authors declare that they did not use generative AI to come up with new ideas for their review.

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2. 

   PREreview

   Oct 27, 2025

   This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/17459808.

   The article "Pre-existing antibodies and age shape the immune response following EV-A71 vaccine: a prospective serological study among the Chinese pediatric population" examines how age and baseline immunity influence the immune response to the EV-A71 vaccine in children. The study uses serological data from 1,585 children aged 6 to 35 months in China who received two doses of the vaccine, and antibody titers were measured before and after immunization. The authors conclude that both age and pre-existing antibodies significantly affect immune responses following vaccination, and they suggest that children should be vaccinated early in life to maximize protection. The manuscript is …

   This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/17459808.

   The article "Pre-existing antibodies and age shape the immune response following EV-A71 vaccine: a prospective serological study among the Chinese pediatric population" examines how age and baseline immunity influence the immune response to the EV-A71 vaccine in children. The study uses serological data from 1,585 children aged 6 to 35 months in China who received two doses of the vaccine, and antibody titers were measured before and after immunization. The authors conclude that both age and pre-existing antibodies significantly affect immune responses following vaccination, and they suggest that children should be vaccinated early in life to maximize protection. The manuscript is well-organized, clearly written, and addresses an important public health topic, as EV-A71 remains a leading cause of severe hand, foot, and mouth disease in young children. However, while the study is scientifically relevant and uses a large dataset, there are major methodological issues and interpretational flaws that must be addressed before the conclusions can be considered valid.

   A major problem with this study is that it does not control for natural EV-A71 infection during the study period. Since EV-A71 is endemic in China and often spreads among children through asymptomatic infection, some participants may have naturally acquired EV-A71 during the follow-up period. Because the study did not monitor participants for infection symptoms or test for viral exposure between vaccine doses, it is impossible to know whether antibody increases were caused by the vaccine or by natural infection. This is a serious confounding variable that weakens the study's internal validity. The authors briefly acknowledge cross-reactivity and natural exposure as limitations but do not attempt to estimate or adjust for this effect. Including regional infection data or conducting sensitivity analyses would make the findings more credible.

   Another major issue is the combination of two fundamentally different populations in the analysis: children from a Phase III clinical trial and children from an observational cohort study. These two groups differ in important ways, such as age, potential exposure to infection, and health monitoring, which introduces bias when the results are pooled together. Although the authors statistically adjust for "data source" in their model, this does not fully correct for structural differences in study design. For example, clinical trial participants were younger and had lower baseline antibody levels than cohort children, who were older and more likely to be naturally exposed to EV-A71 before vaccination. Because of this, vaccine responses might appear different between the two groups for reasons unrelated to age or immune history. The authors should have analyzed these groups separately to show whether the trends they observed were consistent in both populations.

   A third significant weakness in the paper is the authors' interpretation of the fold increase in antibody titers. They conclude that children with higher levels of pre-existing antibodies have weaker vaccine responses because their immune systems are already primed. While a lower fold increase in seropositive children is expected, this result does not necessarily mean that pre-existing antibodies inhibit vaccine response. Instead, it may reflect a natural "ceiling effect," where individuals with already high antibody levels have limited ability to increase further. Another possible explanation is technical; neutralizing antibody assays tend to be less precise at very high titers, compressing the range of detectable increases. The authors do not discuss these alternative explanations, which makes their conclusion feel overstated. They imply a biological mechanism that they did not test experimentally.

   In addition to these major problems, the paper has several minor weaknesses. Some figures lack clear axis labels and statistical descriptions, especially Figures 2 and 3. The authors also do not fully justify their choice of seropositivity cutoffs for classifying antibody levels, which weakens their claims about immune categories. The description of statistical models in the Methods section lacks detail, and it is unclear how model assumptions were checked. Finally, the conclusion states that children should be vaccinated "as early as possible," yet the data show that older children produced stronger antibody responses. This contradiction should be addressed by clarifying whether early vaccination is recommended for protection before exposure, rather than for enhanced immunogenicity.

   In conclusion, this study provides valuable insights into EV-A71 serology in children and highlights a significant relationship between age, pre-existing immunity, and vaccine response. However, the lack of control for natural infection, the flawed pooling of two distinct study populations, and the overinterpretation of immunological findings significantly weaken the reliability of the conclusions. With major revisions that address these methodological and interpretational issues, the study has potential for publication. At its current stage, it requires substantial improvement to ensure scientific accuracy and credibility.

   Competing interests

   The author declares that they have no competing interests.

   Use of Artificial Intelligence (AI)

   The author declares that they used generative AI to come up with new ideas for their review.

   Read the original source
3. 

   Version published to 10.1101/2025.09.17.25335968 on medRxiv

   Sep 17, 2025