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We investigated whether maternal and child peripheral blood immune markers relate to early brain metabolite profiles in children with and without HIV exposure from a South African birth cohort. Methods Within the Drakenstein Child Health Study, a neuroimaging subgroup of children underwent single-voxel magnetic resonance spectroscopy at 2\u20133 years (n=156; 66 HEU, 90 HIV-unexposed). A panel of eighteen immune markers was quantified in blood serum of pregnant women and in their children at 7 weeks and 2 years follow-up. Neurometabolite ratios to creatine were quantified in midline parietal grey matter and left and right parietal white matter. Cross-sectional associations between immune markers and neurometabolite ratios were tested using linear models with robust standard errors, adjusting for age at scan, sex, and voxel tissue composition, and controlling for false discovery rate. Results In children who were HEU, higher concentrations of maternal pro-inflammatory cytokines IL-5 (\u03b2=0.79, p=0.005) and IL-8 (\u03b2=0.64, p=0.02) were positively associated with myo-inositol ratios in midline parietal grey and right parietal white matter regions, respectively. At two years, higher child serum MMP-9 was positively associated with myo-inositol ratios in midline parietal grey matter (\u03b2=1.30, p=0.03). Maternal IL-13 was positively associated with glutamate ratios in the midline parietal grey matter of HIV-unexposed peers (\u03b2=0.42, p&lt;0.0001), with no association in children who are HEU. Conclusions In this South African cohort, HIV exposure-specific associations were observed between certain mother and child immune markers and child neurometabolite ratios at 2\u20133 years. Larger, longitudinal neuroimaging studies integrating neurodevelopmental outcomes are needed to clarify mechanisms and clinical implications.<\/ns3:p>","DOI":"10.12688\/wellcomeopenres.24911.2","type":"journal-article","created":{"date-parts":[[2026,3,9]],"date-time":"2026-03-09T05:09:07Z","timestamp":1773032947000},"page":"611","update-policy":"https:\/\/doi.org\/10.12688\/wellcomeopenres.crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Maternal and child immune profiles are associated with neurometabolite measures of early-life neuroinflammation in children who are HIV-exposed and uninfected: a South African birth cohort"],"prefix":"10.12688","volume":"10","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9286-9595","authenticated-orcid":false,"given":"Cesc","family":"Bertran-Cobo","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Frances C.","family":"Robertson","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2638-6725","authenticated-orcid":false,"given":"Tusekile Sarah","family":"Kangwa","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9345-9309","authenticated-orcid":false,"given":"Jenna","family":"Annandale","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sivenesi","family":"Subramoney","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Katherine L.","family":"Narr","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shantanu H.","family":"Joshi","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7269-1416","authenticated-orcid":false,"given":"Nadia","family":"Hoffman","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9046-759X","authenticated-orcid":false,"given":"Heather J.","family":"Zar","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7218-7810","authenticated-orcid":false,"given":"Dan J.","family":"Stein","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0276-9660","authenticated-orcid":false,"given":"Kirsten A.","family":"Donald","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4887-1644","authenticated-orcid":false,"given":"Catherine J.","family":"Wedderburn","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Petrus J. 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Research"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/wellcomeopenresearch.org\/articles\/10-611\/v2\/xml","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/wellcomeopenresearch.org\/articles\/10-611\/v2\/pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/wellcomeopenresearch.org\/articles\/10-611\/v2\/iparadigms","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,3,12]],"date-time":"2026-03-12T08:04:18Z","timestamp":1773302658000},"score":1,"resource":{"primary":{"URL":"https:\/\/wellcomeopenresearch.org\/articles\/10-611\/v2"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,3,9]]},"references-count":79,"URL":"https:\/\/doi.org\/10.12688\/wellcomeopenres.24911.2","relation":{"has-version":[{"id-type":"doi","id":"10.12688\/wellcomeopenres.24911.1","asserted-by":"subject"}],"has-review":[{"id-type":"doi","id":"10.21956\/wellcomeopenres.28715.r150235","asserted-by":"subject"},{"id-type":"doi","id":"10.21956\/wellcomeopenres.28715.r150508","asserted-by":"object"},{"id-type":"doi","id":"10.21956\/wellcomeopenres.28715.r150234","asserted-by":"object"},{"id-type":"doi","id":"10.21956\/wellcomeopenres.28715.r150236","asserted-by":"object"},{"id-type":"doi","id":"10.21956\/wellcomeopenres.28715.r150235","asserted-by":"object"}]},"ISSN":["2398-502X"],"issn-type":[{"value":"2398-502X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,3,9]]},"assertion":[{"value":"Indexed","URL":"https:\/\/wellcomeopenresearch.org\/articles\/10-611\/v2#article-reports","order":0,"name":"referee-status","label":"Referee status","group":{"name":"current-referee-status","label":"Current Referee Status"}},{"value":"10.21956\/wellcomeopenres.28715.r150235, Kim Cecil, Cincinnati Children's Hospital Medical Center, Cincinnati, 12 Mar 2026, version 2, indexed","URL":"https:\/\/wellcomeopenresearch.org\/articles\/10-611\/v2#referee-response-150235","order":0,"name":"referee-response-150235","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"10.21956\/wellcomeopenres.27441.r138485, Jaime H Vera, Brighton and Sussex Medical School, Brighton, 11 Dec 2025, version 1, 2 approved with reservations, 1 not approved","URL":"https:\/\/wellcomeopenresearch.org\/articles\/10-611\/v1#referee-response-138485","order":1,"name":"referee-response-138485","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"<b>Cesc Bertran Cobo<\/b>; \n<i>Posted: 12 Feb 2026<\/i>; \n<i><b>Reviewer <\/b>\u2013 Overall Impression: &quot;This is an important and well-designed study. The paper presents unique longitudinal immune data paired with MRS outcomes in a South African HEU cohort something rarely available. However, several aspects need clarification to avoid overstating conclusions and to ensure the interpretation matches the strength of the results.&quot;<\/i> &nbsp; \n<b>Response <\/b>\u2013 We thank the reviewer for their careful reading of the manuscript and for the constructive suggestions to better align our interpretation with the strength of the evidence. We have clarified the role of contextual factors and use more cautious language to strengthen the paper. &nbsp; \n<i><b>Reviewer <\/b>\u2013 1. Contextualisation of prior literature: &quot;The introduction summarises well the inconsistent findings across geographical regions. I suggest explicitly highlighting that these discrepancies may also reflect differences in social determinants of health, environmental exposures, and co-infections, which can shape immune maturation. This helps the reader understand that immune differences in HEU children are not solely attributable to HIV exposure.&quot;<\/i> &nbsp; \n<b>Response <\/b>\u2013 We agree and have expanded the Introduction to explicitly note that heterogeneity in immune findings in children who are HEU may reflect social determinants of health, environmental exposures, and co-infections that may shape early immune maturation. &nbsp; We have added text highlighting these contextual influences and include supporting references: \u201cThese inconsistencies may reflect differences in regional exposure to co-infections, social determinants of health, and early-life environmental factors that influence immune maturation\n<sup>A,B<\/sup>. They also highlight the need for further research to elucidate how HIV exposure affects immune and neurodevelopmental trajectories in children across different social and geographical contexts.\u201d &nbsp; A. Kuhlman KR, Horn SR, Chiang JJ, Bower JE. Early life adversity exposure and circulating markers of inflammation in children and adolescents: A systematic review and meta-analysis. Brain Behav Immun. 2020;86:30-42. doi: 10.1016\/j.bbi.2019.04.028. &nbsp; B. Gilman SE, Hornig M, Ghassabian A, et al. Socioeconomic disadvantage, gestational immune activity, and neurodevelopment in early childhood. Proc Natl Acad Sci USA. 2017;114(26):6728-6733. doi: 10.1073\/pnas.1617698114. &nbsp;  \n<i><b>Reviewer<\/b> \u2013 2. Over-interpretation of immune brain associations. &quot;Only a subset of immune markers (e.g., IL-5, IL-8, MMP-9, NGAL) showed significant associations. Many initial differences did not survive multiple-comparison correction. Statements implying widespread associations should be softened to reflect that the findings relate to specific markers, not broad immune dysregulation.&quot;<\/i> &nbsp; \n<b>Response <\/b>\u2013 Thank you. We have revised the Discussion to emphasise that associations were identified for specific serum markers and that many differences did not survive multiple comparison correction. We softened wording that could imply widespread immune dysregulation and reframed the findings as selective neuroimmune associations that differ by HIV exposure group. &nbsp; Examples of rewording include:  Abstract conclusions: \u201cIn this South African cohort, HIV exposure-specific associations were observed between certain mother and child immune markers and child neurometabolite ratios at 2\u20133 years.\u201d Line 381 (Discussion, 3rd paragraph): \u201cTogether, these findings support our hypothesis that specific maternal and child immune markers are associated with early-life elevated myo-inositol in the parietal white matter of children who are HEU.\u201d Lines 386\u2013390 (Discussion, 3rd paragraph): \u201cMyo-inositol is clinically recognized as a marker of neuroinflammation and gliosis, and it has been linked to cognitive impairment in children living with HIV. Thus, increases in myo-inositol alterations in children who are HEU may potentially result from maternal immune dysregulation during pregnancy. IL-8 is a plausible candidate in this context, given its role as a pro-inflammatory chemokine in perinatal immune activation and, if dysregulated, its potential negative effects on brain maturation and neurodevelopment. Line 465 (Discussion, last paragraph): \u201cIn conclusion, this study suggests that maternal HIV status is associated with selective distinct alterations in maternal and child immune profiles, and with exposure-specific associations between a subset of maternal\/child serum markers and child neurometabolite ratios at 2\u20133 years.\u201d  &nbsp;\n<i><b>Reviewer <\/b>\u2013 3. Limited adjustment for social determinants: &quot;The manuscript does not adjust for social determinants of health despite clear group differences. These factors, which strongly influence inflammation and neurodevelopment, likely contribute to observed differences. The authors should acknowledge this in the limitations.&quot;<\/i> &nbsp; \n<b>Response <\/b>\u2013 We clarified that key socioeconomic variables were comparable between HEU and HU groups in this subset, and that we conducted sensitivity analyses for measured variables that did differ between groups. We agree that residual confounding remains possible and have expanded the limitations to acknowledge that we could not account for additional factors that may influence immune development and neurometabolite associations. &nbsp; Wording now reads as follows (line 445, Discussion section, Strengths and limitations paragraph): \u201cWhile socioeconomic status was similar across children who were HEU and HU, maternal age at delivery, as well as rates of depression and alcohol use during pregnancy differed between groups. Reassuringly, our results held after correcting for these variables in sensitivity analyses, although other unmeasured factors may contribute to neuroimmune variability.\u201d &nbsp; \n<i><b>Reviewer <\/b>\u2013 4. Interpretation of non-significant infant\/child immune differences: &quot;Most differences in infant and child serum markers disappear after correction. This should be stated clearly, emphasising that the data are more consistent with normalisation of markers over time rather than persistent dysregulation.&quot;<\/i> &nbsp; \n<b>Response <\/b>\u2013 We agree and edited the Discussion to more explicitly state that, after correction, group differences were primarily observed during pregnancy and that infant\/child differences were attenuated, which is more consistent with partial normalisation of peripheral markers over time than persistent dysregulation across early life. &nbsp; Wording now reads as follows (line 374, Discussion section, end of 2nd paragraph): \u201cDespite this, our longitudinal analysis from six weeks to two years did not reveal significant differences in serum marker trajectories between HEU and HU children. This may be consistent with partial normalisation rather than persistent differences of peripheral markers over time in early childhood.\u201d &nbsp; \n<i><b>Reviewer <\/b>\u2013 5. Mediation analysis interpretation: &quot;The mediation models did not show evidence that serum markers explain the effect of maternal HIV on neurometabolites. This may reflect small sample size, weak group differences, unmeasured social determinants, or that immune pathways may not be the primary mechanism.&quot;<\/i> &nbsp; \n<b>Response <\/b>\u2013 Thank you, we agree and have reviewed the mediation paragraph in the Discussion to consider the interpretation of the mediation results. We have added potential explanations including limited power in complete-case mediation models, small indirect effects, attenuation of child marker differences after correction, residual confounding by unmeasured social\/environmental factors, and others. We consider that immune pathways may not be primary mechanisms but equally, the results do not exclude immune mechanisms but suggest that no single measured marker explained the maternal HIV\u2013neurometabolite association in isolation. &nbsp; Wording now reads as follows (line 413, Discussion section, 5th paragraph): \u201cOur mediation analyses did not identify any individual serum marker that mediated the association between maternal HIV status and child neurometabolite ratios at 2\u20133 years. This null result should be interpreted cautiously given limited power to detect indirect effects in our complete-case with paired immune and MRS data. Moreover, group differences in several serum markers were modest and further attenuated after multiple-comparison correction, reducing power to detect mediation by any single marker. Residual confounding by social and environmental determinants, as well as biological pathways (including antiretroviral exposure and co-infections), may also play a role. Taken together, these findings do not rule out immune mechanisms but suggest that no single measured marker explains the maternal HIV\u2013neurometabolite association in isolation.\u201d &nbsp; \n<i><b>Reviewer <\/b>\u2013 6. Final conclusion too strong: &quot;The conclusion should use more cautious language (\u201csuggests\u201d, \u201cindicates\u201d, \u201cprovides preliminary evidence\u201d) rather than \u201cdemonstrates\u201d, given the exploratory nature of the findings and potential confounding.&quot;<\/i> &nbsp; \n<b>Response<\/b> \u2013&nbsp;We have adopted more cautious language as suggested and emphasise the exploratory nature of the findings and the need for larger, longitudinal studies integrating neurodevelopmental outcomes and contextual determinants. Wording now reads as follows (line 465, Discussion, last paragraph): \u201cIn conclusion, this study suggests that maternal HIV status is associated with selective distinct alterations in maternal and child immune profiles, and with exposure-specific associations between a subset of maternal\/child serum markers and child neurometabolite ratios at 2\u20133 years.\u201d","URL":"https:\/\/wellcomeopenresearch.org\/articles\/10-611\/v2#referee-comment-6431","order":2,"name":"referee-comment-6431","label":"Referee Comment","group":{"name":"article-reports","label":"Article Reports"}},{"value":"10.21956\/wellcomeopenres.27441.r139380, Ashley Harris, Department of Radiology, University of Calgary, Calgary, 24 Dec 2025, version 1, 2 approved with reservations, 1 not approved","URL":"https:\/\/wellcomeopenresearch.org\/articles\/10-611\/v1#referee-response-139380","order":3,"name":"referee-response-139380","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"<b>Cesc Bertran Cobo<\/b>; \n<i>Posted: 12 Feb 2026<\/i>; \n<i><b>Reviewer \u2013 <\/b>Overall Impression: &quot;I think this is a well executed study with an impressive amount of data. I have several comments but they are all minor and intended to provide clarity as I expect there audience will be broad (clinicians and researchers with or without expertise in child development, blood serum analyses and spectroscopy).&nbsp; Reviewing the previous reviewer comments \u2013 I do not see how the choline and NAA exclusions were adjusted. There is no comment about these data being excluded so perhaps they have just been included for completeness? Please clarify. <b>&quot;<\/b><\/i>\n<b> Response<\/b> \u2013 We thank the reviewer for the thoughtful and constructive feedback and for emphasising clarity for a broad audience spanning clinicians and researchers with varied expertise. Regarding choline and NAA adjustments, we apologize for the confusion caused by our earlier wording. As outlined in the Reviewer #1 response, choline and NAA were not excluded from statistical testing; they were analysed in the same models as other metabolites with voxel tissue fractions included a priori as covariates, highlighting their sensitivity to tissue composition in adjusted models. \n<i><b>Reviewer <\/b>\u2013&nbsp;Other comments: 1.&nbsp; &nbsp; Introduction 1.1.&nbsp; &nbsp; Is there a citation for the DCHS study as a whole? Would be nice to include<\/i> Response \u2013&nbsp;We have added citations for the DCHS cohort overview at first mention in the Introduction section (line 25), in addition to the existing protocol citations in Methods. \n<i>Reviewer \u2013 1.2.&nbsp; &nbsp; Final paragraph could be more clear as I\u2019m confused about what is novel and what the sampling is \u2013 specifically have already published the MRS data (correct?) so the addition is longitudinal blood data or is there longitudinal MRS data as well?<\/i> \n<b>Response<\/b> \u2013 Thank you, we have reviewed the Introduction paragraph for clarity. Wording now reads as follows (line 55, Introduction section, last paragraph): \u201cOur study aimed to address this gap by investigating maternal and child peripheral blood immune profiles from pregnancy through birth and early childhood, and their associations with brain metabolite levels at age 2\u20133 years, using existing data from the DCHS cohort of South African children.\u201d \n<i><b>Reviewer \u2013 2.<\/b>&nbsp; &nbsp; Methods 2.1.&nbsp; &nbsp; Reports of participants \u2013 This is confusing as it is partial information. It is all in the results so I would just state that the data is collected as apart of the DCHS study and then report the data that is used in the results as this is more appropriate and it is clearly written\/understood in the results section right now.<\/i> \n<b>Response<\/b> \u2013&nbsp;Thank you. We have retained a concise Methods description (with detailed protocol references) and added a signposting sentence indicating that final analytic sample sizes by timepoint and modality are reported in the Results and Supplementary Figure 1. Wording now reads as follows (line 88, Methods section, end of 2nd paragraph): \u201cFinal sample sizes contributing to each analysis are reported in the Results and Supplementary Figure 1.\u201d \n<i><b>Reviewer \u2013 2.2<\/b>.&nbsp; &nbsp; HIV status section doesn\u2019t seem to fit well in the sociodemographic methods \u2013 perhaps would be best in own section<\/i> \n<b>Response <\/b>\u2013&nbsp;Thank you. We agree and have created a new sub-section within the Methods section, called \u201cMaternal and child HIV status\u201d (line 88, in Methods section). \n<i><b>Reviewer \u2013 2.3<\/b>.&nbsp; &nbsp; Never stated what sociodemographic information was collected<\/i> \n<b>Response \u2013&nbsp;<\/b>We have added a brief sentence listing the main sociodemographic and psychosocial domains collected (income, education\/employment, relationship status, smoking\/alcohol, depression, infant feeding and growth), including references to the detailed DCHS protocol publications. Wording now reads as follows (line 102, Methods section, Sociodemographic data collection sub-section): \u201cVariables included household income, maternal education and employment, relationship status, smoking and alcohol use during pregnancy, depressive symptoms, birth outcomes, infant feeding, and child growth indicators.\u201d \n<i><b>Reviewer \u2013 2.4.<\/b>&nbsp; &nbsp; What is GM-CSF? I assume IL-XX is interleukin-XX, I don\u2019t know what INF-gamma is either (I do recognize these are standard assay to those who do these blood biomarkers but have targeted a diverse audience with this manuscript), similarly fill name of MMP-9, NAGL, YKL-40 is likely appropriate and the brief descriptions are appreciated.<\/i> \n<b>Response <\/b>\u2013We agree and have included IFN-\u03b3 notation and defining GM-CSF, IFN-\u03b3, and TNF\u03b1 at first use. We also note that a comprehensive marker table (names\/abbreviations and relevant literature) is provided in the Supplementary Files, and we have enquired with the editors about displaying this table in the main text, as we believe this addition provides clarification for broader audiences. Wording now reads as follows (line 114, Methods section, Immune assays sub-section): \u201cConcentrations of granulocyte\u2013macrophage colony-stimulating factor (GM-CSF), interferon gamma INF(IFN-\u03b3), interleukins IL-1\u03b2, IL-2, IL-5, IL-6, IL-7, IL-8 (CXCL8), tumour necrosis factor alpha (TNF-\u03b1), IL-4, IL-10, IL-12p70, and IL-13 were measured using a Milliplex &reg; Luminex 13-plex kit (#HSTCMAG28SPMX13; Merck) according to the manufacturer\u2019s instructions.\u201d \n<i><b>Reviewer \u2013 2.5<\/b>.&nbsp; &nbsp; Would be beneficial to quantify relative to water and perform full quantification (see Near et al 2020) if have unsuppressed water data as creatine may be changing, if you don\u2019t have water data for quantification, I think this should be acknowledged.<\/i> \n<b>Response <\/b>\u2013 Unsuppressed water reference scans were not available, therefore, we report metabolite ratios to total creatine and not absolute concentrations. Wording now reads as follows (line 150, Methods section, MRS data processing sub-section): \u201cUnsuppressed water reference scans were not available, therefore, we do not report neurometabolite absolute concentrations. \n<b>\u201d <\/b>\n<i><b>Reviewer <\/b>\u2013 2.6.&nbsp; &nbsp; Units for FWHM missing and the metabolite used or the SNR and FWHM for quality assessment<\/i> \n<b>Response \u2013 <\/b>We clarified the FWHM units (ppm) and the QC approach. Wording now reads as follows (line 150, Methods section, MRS data processing sub-section): \u201cSpectral quality was visually inspected and assessed using signal-to-noise ratio (SNR) and full width at half maximum (FWHM, in ppm) given by LCModel. Spectra with FWHM values greater than 0.08 or SNR values lower than 10 were considered of low quality and therefore excluded.\u201d \n<i><b>Reviewer \u2013 3.<\/b>&nbsp; &nbsp; Results 3.1.&nbsp; &nbsp; The description of the participants in the results is much clearer \u2013 suggest just reporting the metrics acquired in the methods<\/i> \n<b>Response \u2013<\/b>&nbsp;Thank you, we proceeded as suggested. Wording now reads as follows (line 102, Methods section, Sociodemographic data collection sub-section): \u201cVariables included household income, maternal education and employment, relationship status, smoking and alcohol use during pregnancy, depressive symptoms, birth outcomes, infant feeding, and child growth indicators. \n<b>\u201d<\/b>\n<i><b>Reviewer \u2013 3.2<\/b>.&nbsp; &nbsp; I cannot figure out how to get the supplemental figures so have not examined. From the text they appear to be appropriate.<\/i> \n<b>Response <\/b>\u2013&nbsp;Please see line 495 (Data availability section, Extended data sub-section). We mention there that Supplementary Figures are contained within the \u201cSupplementary Files (PDF)\u201d deposited in the OSF project linked by DOI. \n<i><b>Reviewer \u2013 3.3.<\/b>&nbsp; &nbsp; Figure 1 \u2013 very interesting results - the font on the axes (even in the downloaded ppt) is too small. Can see zoomed in on pdf but recommended fixing<\/i> \n<b>Response \u2013<\/b>&nbsp;Thank you. We have corrected Figure 1 by increasing axis font sizes for readability and have submitted it to the Editorial Team to have it replaced in the main text. \n<i><b>Reviewer \u2013 3.4<\/b>.&nbsp; &nbsp; If I understand, a limited correction for multiple comparisons was performed \u2013 only corrected for number that were significant in the primary analysis, not corrected for all comparisons performed.<\/i> \n<b>Response \u2013<\/b>&nbsp;We clarified that Benjamini\u2013Hochberg correction was applied across all tests within each analysis set, irrespective of unadjusted p-values. Wording now reads as follows (line 195, Methods section, Statistical analysis sub-section, last paragraph): \u201cResults were considered significant at a p-value threshold of &lt; 0.05 (two-tailed). BH correction was applied across all tests within each analysis set. \n<b>\u201d<\/b>\n<i><b>Reviewer \u2013 3.5<\/b>.&nbsp; &nbsp; Given serum markers didn\u2019t change but have different results in the 6 week and 2 year data \u2013 this raises a suspicion that should be acknowledged but I don\u2019t see as a fatal flaw<\/i> \n<b>Response \u2013 <\/b>Previous studies, as well as our findings, demonstrate increases in cytokine levels during the first two years of life, with subsequent stabilisation, consistent with immune system maturation in early life.  Ray JE, Dobbs KR, Ogolla SO, et al. Clinical and Immunological Outcomes of HIV-Exposed Uninfected and HIV-Unexposed Uninfected Children in the First 24 Months of Life in Western Kenya. BMC Infectious Diseases (2023) (1):156. doi: 10.1186\/s12879-024-09051-3.&nbsp; Miyamoto M, Gouv&ecirc;a AFTB, Ono E, et al. Immune development in HIV-exposed uninfected children born to HIV-infected women. Rev Inst Med Trop Sao Paulo (2017) 59:e30. doi: 10.1590\/S1678-9946201759030.&nbsp;  \n<i><b>Reviewer \u2013 4.<\/b>&nbsp; &nbsp; Discussion 4.1.&nbsp; &nbsp; Discussion is appropriate but largely a summary of the results with a little context. I think it would be compelling to interpret and speculate on the data more \u2013 does this give direction for clinicians now? Should actions be taken to consider the immune profiles and perhaps brain development trajectories for these children?<\/i> \n<b>Response <\/b>\u2013&nbsp;We strengthened the Discussion by adding a paragraph on clinical insights and next steps, emphasizing that findings are exploratory, but that they support the importance of optimising maternal health during pregnancy. Wording now reads as follows (line 466, Discussion section, last paragraph): \u201cIn conclusion, this study suggests that maternal HIV status is associated with selective alterations in maternal and child immune profiles, and with exposure-specific associations between a subset of maternal\/child serum markers and child neurometabolite ratios at 2\u20133 years. These findings provide preliminary evidence that perinatal HIV exposure may be followed by altered early-life neuroimmune coupling, although associations are marker-specific and require replication. Clinically, our results reinforce the importance of optimising maternal HIV health during pregnancy, with early ART initiation and monitoring and viral suppression to minimise immune activation, as well as prevention and management of inflammatory comorbidities. Future studies should confirm our findings in larger cohorts, integrate more detailed social and environmental determinants, and link perinatal immune profiles to longitudinal neurodevelopmental trajectories to clarify mechanisms and identify modifiable pathways that support optimal development in children who are HEU.\u201d","URL":"https:\/\/wellcomeopenresearch.org\/articles\/10-611\/v2#referee-comment-6432","order":4,"name":"referee-comment-6432","label":"Referee Comment","group":{"name":"article-reports","label":"Article Reports"}},{"value":"This work was supported by Wellcome [222020, 221372, 203525]; National Research Foundation (NRF); National Institutes of Health (NIH) [U01AI110466-01A1]; Gates Foundation [OPP1017641, OPP1017579]; South African Medical Research Council (SAMRC); Academy of Medical Sciences Newton Advanced Fellowship, UK Government\u2019s Newton Fund [NAF002\/1001]; National Institute on Alcohol Abuse and Alcoholism (NIAAA) [R21AA023887]; Collaborative Initiative on Fetal Alcohol Spectrum Disorders (CIFASD) [U24 AA014811]; US Brain and Behavior Foundation Independent Investigator grant [24467]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.","order":5,"name":"grant-information","label":"Grant Information"},{"value":"This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.","order":0,"name":"copyright-info","label":"Copyright"}]}}