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. 2025 Jul 2;20(7):e0321982. doi: 10.1371/journal.pone.0321982

Measles vaccines and non-specific effects on mortality or morbidity: A systematic review and meta-analysis

Louise A Fournais 1,2,*,#, Anne C Zimakoff 1,#, Andreas Jensen 1,, Jeppe H Svanholm 3,4,, Ingvild Fosse 3,5,, Lone G Stensballe 1,3,
Editor: Ahmad Khalid Aalemi6
PMCID: PMC12221017  PMID: 40601595

Abstract

Background

This review evaluates the hypothesis of beneficial non-specific effects of the standard-titre measles vaccine.

Methods

We conducted a systematic review and meta-analysis of randomised controlled trials. Trials included standard or high-titre live attenuated measles containing vaccines compared to other vaccines or placebo. The primary outcomes were mortality and morbidity. Secondary outcomes were infections, antibiotic use, atopy, allergies, asthma, and atopic dermatitis.

Findings

23 articles were included in this systematic review.

Mortality: Two doses of measles vaccine vs. only one dose showed no significant effect on mortality; risk ratio (RR) = 1.05 (95% CI: 0.78 to 1.41), p = 0.76. The analysis was based on a relative risk reduction (RRR) of 25% and a control group event rate of 2.32% as measured in the actual trials included in the analysis. In males, the association was rejected: RR = 1.09 (0.86 to 1.37), p = 0.47. In females, the association was not rejected at 25%,but was at 33% level: RR = 1.0 (0.64 to 1.54), p = 0.99.

Morbidity: Overall, the hypothesis was rejected: RR = 0.94 (0.80 to 1.10), p = 0.43. The rejection was sustained for both sexes: females RR = 0.95 (0.77 to 1.18), p = 0.6; males RR = 0.92 (0.83 to 1.03), p = 0.13.

Interpretation

Based on evidence from randomised controlled trials, this systematic literature review and meta-analysis did not support the hypothesis of non-specific effects of standard-titre measles containing vaccines. Trial Sequential Analysis indicated that the meta-analysis included sufficient data to reach this conclusion.

Trial registration

PROSPERO CRD42022344473

Introduction

Measles is an acute viral disease causing about 134,200 deaths yearly [1]. Measles vaccines are effective; but due to the contagiousness of the virus, ≥ 95% must be vaccinated to control transmission [2]. First doses are usually given at 12 months of age, or from 9 months in endemic settings [3].

In the late 1970s, observational studies in low-income countries revealed an unexpected decline in overall child mortality among measles-vaccinated children that surpassed the anticipated reduction in measles-related deaths [4]. This led to the hypothesis that live-attenuated measles containing vaccines (MCVs) have beneficial non-specific effects. Subsequently randomised controlled trials (RCTs) tested the safety and efficacy of standard-titre (STMV) and high-titre measles-containing vaccines (HTMV) in Senegal, Haiti, Sudan, and Guinea Bissau in the late 1980s and 1990s [58]. These trials showed increased long-term mortality, especially in females, after HTMV [9]. Consequently, HTMVs were withdrawn from child immunisation programmes in 1992 [10].

Non-specific effects of vaccines are defined as health effects reaching beyond protection against the target pathogen. The hypothesis of beneficial non-specific effects of MCVs has been investigated for more than 30 years. Prior studies suggested that live-attenuated vaccines may reduce child mortality, morbidity and improve growth [1113], while inactivated vaccines may worsen these outcomes [14,15]. While numerous non-randomised studies proposed such effects [13,14], statistically significant results have rarely been replicated in RCTs [11,12,16]. Thus, the existing results were ambiguous. Since MCVs are used globally in an era where vaccine hesitancy is recognized as a major global health threat [17], it is crucial to understand health effects of vaccines and clinically important to know whether the non-specific effects observed on observational studies were attributable to confounding.

This systematic review summarises, meta-analyses, and evaluate evidence on potential non-specific effects of MCVs. Focus is on morbidity and mortality after STMV, though HTMV is also considered.

Methods

This systematic review followed the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) statement and The Cochrane Handbook [18,19]. The protocol was registered in the International prospective register of systematic reviews (PROSPERO, CRD42022344473). As RCTs represent the highest level of evidence [18], only RCTs were included. Ethical approval was not needed since all data included in this review has already been published in other articles that had gained consent.

The co-primary outcomes were mortality and morbidity. Secondary analyses scrutinised sex-differential effects and focused on the follow-up period where infants who were randomised to early MCV were compared with control-infants not yet vaccinated with MCV (one vs. zero dose MCV).

Systematic search and screening

A systematic search was initiated in February 2022. The search was computerised and performed on EMBASE and PubMed. The search was repeated weekly until October 2022 and confirmed in August 2023. The search string can be found in supplementary materials (S1 Table). EndNote 20 and Covidence were used to manage the identified articles. Covidence was used for the screening process. Two authors screened abstracts independently according to the PICO criteria (S1 Appendix). Discrepancies were resolved by a third author. The three authors independently screened all full texts. Disagreements were resolved by group discussion or with the senior researcher.

Quality assessment and data extraction

Three authors read all included trials and performed independent quality assessment and data extraction. Disagreements were discussed in the group. The quality assessment was based on the ROB-2 tool, as recommended by the Cochrane Handbook [20]. (S2 Table and S1 Appendix)

Statistics

Two different meta-analyses were performed: forest plots using R version 4.2.1 (R Core Team, Vienna, Austria) with fixed and random effects, and Trial Sequential Analyses (TSA) using the TSA software, 0.9.5.10 beta version (available from ctu.dk/tsa) [21]. TSA helps determine when enough data have been collected to draw conclusions. To avoid including the same child more than once, only data from original trials were used in the meta-analyses. The methods are further detailed in S1 Appendix.

Results

The literature search identified 4315 unique articles. After initial screening, 4224 articles were excluded due to non-eligible interventions or study design. The full text of 92 articles were assessed, and 23 were included. Reasons for exclusions are presented in the PRISMA diagram (Fig 1) and in S5 Table. Trial characteristics are presented in supplementary materials (S1 Fig and S2 Table-S4 Table).

Fig 1. PRISMA.

Fig 1

Flowchart of the selection process.

The standard-titre measles vaccine

Mortality.

Seven trials (35,516 participants) investigated mortality as the primary outcome. Five were original trials, two were re-analyses of already included trials. Four trials compared a two-dose schedule with a one-dose schedule. Three trials compared a dose at 9 months with an additional early dose at 4 months. Follow-up was until 36 months (two trials) [11,12] or 60 months (one trial) [16]. One trial compared a dose at 9 months with a booster dose at 18 months, with 48 months follow-up [22]. The fifth original trial was cluster randomised and compared a ’restrictive measles vaccine policy‘ with a ’measles vaccine for all policy‘ with 60 months follow-up [23]. Two re-analyses used data from the trials with an extra early dose and 36 months of follow-up [24,25].

Forest plots showed no differences in mortality: crude fixed effect model RR = 0·99 (95% confidence interval 0·85 to 1·16). Random effects model: RR = 1·00 (0·82 to 1·22), I2 = 37% (S2 Fig). Adjusted fixed effect model: RR = 1·00 (0·84 to 1·18). Adjusted random effects model: RR = 1·00 (0·83 to 1·20), I2 = 7% (Fig 2).

Fig 2. Forest plot: Mortality.

Fig 2

Forest plot of adjusted mortality estimates from original trials. Results shown for both fixed effect and random effects models.

TSA included crude estimates from three original trials [11,12,16] comparing two-dose and one-dose measles vaccine schedules. Two trials were excluded due to non-comparable designs. One trial [23] used a ‘MCV-for-all-policy’ versus a ‘restricted MCV. The other trial [22] used a composite outcome of both admissions and mortality. When comparing two vs. one MCV dose, the TSA showed that enough data was collected to conclusively reject a 25% relative reduction in the mortality risk, pooled effect RR = 1·05 (0·78 to 1·41), p = 0·76 (Fig 3). This is shown by the z-curve (Fig 3, green line). The z-curve falls below the 97·5 percentile of the standard normal distribution of 1·96 (Fig 3, blue dashed line), indicating no significant difference. The confidence interval and p-value lead to the same conclusion. Furthermore, the z-curve reached the area of futility. This is represented by the red triangle. This triangle is calculated by the TSA software and illustrates the point at which sufficient data has been included into the analysis to conclusively reject the hypothesis.

Fig 3. TSA: Mortality.

Fig 3

Pooled Effect: RR = 1·05 (CI: 0·78 to 1·41, p = 0·76), Q = 5·71 (p = 0·06), I2 = 0·65, D2 = 0·67.

One vs zero doses of MCV

Three studies compared infants randomised to early MCV with infants randomised to no early dose as secondary analyses. All trials were original with non-overlapping populations. Mortality was compared after the early extra dose given at 4·5 months of age [11], 4 weeks after the third DTP vaccine [12], and at 4 months of age [16]), and at the routine dose at 9 months. The first trial [11] with follow-up of 4·5 months found mortality rate ratio (MRR) = 0·67 (0·38 to 1·19). The second trial [12] with follow-up from 4 weeks after the third DTP vaccine found MRR = 1·10 (0·66 to1·83). The third trial [16] with follow-up of 5 months found HR = 0·94 (0·45 to 1·96). No trial showed significant differences. Using crude estimates, a forest plot meta-analysis was performed. The fixed effect model resulted in a pooled RR = 0·90 (0·65 to 1·26). The random effects model yielded a pooled RR = 0·90 (0·65 to 1·26), I2 = 0% (Fig A in S2 Appendix). The TSA analysis found no significant difference for RRR = 25%, but more data were needed to reach a conclusive result (Fig B in S2 Appendix). For RRR = 33% (Fig C in S2 Appendix), the z-curve reached the area of futility. Thus, enough data was included to reach the conclusion that no 33% reduction in mortality risk was found when investigating one vs. zero doses of MCV.

Potential sex-differential effects

Sex-specific subgroup analyses were performed. In males, a 25% mortality reduction was conclusively rejected, pooled effect RR = 1·09 (0·86 to 1·37) (Fig A in S3 Appendix). For females, the hypothesis of a 33% mortality reduction when comparing a two-dose programme with one dose was conclusively rejected, pooled effect RR = 1·0 (0·64 to 1·54) (Fig B in S3 Appendix).

Morbidity

Three original trials (18,700 participants) investigated admissions or outpatient consultations as primary outcomes. One trial compared one MCV dose at 9 months with an extra booster dose at 18 months with follow-up until 48 months [22]. Another trial compared an early dose of MCV at 5–7 months with placebo and followed the children until 12 months [26]. The third original trial compared a dose of MCV vaccine with a health check-up and followed the children for 2 months [27]. Six other trials (26,519 participants) investigated these outcomes as secondary. Of these six trials, one was original. This trial compared a ’restrictive measles vaccine policy‘ with a ’measles vaccine for all policy‘ with follow-up until 60 months [23]. The remaining five trials were re-analyses or sub-studies of other trials included in this review [24,2831].

The meta-analysis did not show any significant differences in admission rates between intervention and control groups across five trials with non-overlapping populations. Two forest plots were made using the crude and adjusted (for measles cases, accidental deaths, season, age, and sex) estimates of each trial. One trial was excluded since crude admission data were not published [22]. The pooled RR was 0·91 (0·82 to 1·00) (S3 Fig). Using adjusted estimates, the fixed effects model resulted in a pooled RR = 1·00 (0·92 to 1·08); the random effects model in RR = 1·00 (0·92 to 1·08), I2 = 1% (Fig 4).

Fig 4. Forest plot: Morbidity.

Fig 4

Forest plot based on adjusted estimates from original trials of morbidity. Calculated using fixed and random effects models.

Two trials with comparable interventions [24,31] were included in the TSA. The z-curve reached the area of futility, showing that enough data had been included to conclusively reject a 25% relative reduction in morbidity risk for two doses vs. one. Pooled effect RR = 0·94 (0·80 to 1·10), p = 0·43 (Fig 5).

Fig 5. TSA: Morbidity.

Fig 5

Pooled Effect: RR = 0·94 (C.I: 0·80 to 1·10, p = 0·43), Q = 1·27 (p = 0·26), I2 = 0·21, D2 = 0·24.

One vs zero doses of MCV

Three trials examined the effects of one versus zero doses of MCV. Two trials presented this as the primary analysis. The extra early dose was given at 4 weeks after the third DTP vaccine [24], at 5–7 months [26], and at 4·5 months [29]. Two trials were re-analyses of other RCTs [24, 29] and one was original [26]. No populations overlapped. The first trial reported HR = 1·06 (0·77 to 1·45) [24], the second HR = 1·03 (0·91 to 1·18) [26], and the third HR = 0·78 (0·58 to 1·07) [29]. No significant differences were found. A forest plot using crude fixed effect estimates showed a pooled RR = 1·01 (0·93 to 1·09). The random effects model yielded a pooled RR = 0·93 (0·74 to 1·17), I2 = 68% (Fig A in S4 Appendix). The TSA analysis concluded that no statistical difference was found between the two groups based on a 25% RRR. (Fig B in S4 Appendix).

Potential sex differential effects

A subgroup TSA by sex confirmed that a 25% relative reduction in mortality risk was conclusively rejected for both sexes when comparing two doses to one dose. In females, the pooled effect RR was 0·95 (0·77 to 1·18) (Fig A in S5 Appendix). In males, the pooled effect RR was 0·92 (0·83 to 1·03) (Fig B in S5 Appendix).

The high titre measles vaccine

Eight trials or re-analyses on HTMV were identified [8,9,3237]. One trial found an association between HTMV and a difference in mortality for both sexes. Three trials linked HTMV to increased mortality in female infants only. TSA from five trials [8,32,34,35,38] compared mortality effects of HTMV to STMV for both sexes. The z-curve crossed the boundary of harm (solid red line). Thus, a conclusive and significant negative effect of the HTMV was reproduced with a 25% risk reduction: RR = 1·22 (1·02 to 1·46, p = 0·03). (Fig A in S6 Appendix). The effect was significant in females only, but inconclusive at a 25% risk reduction as the z-curve did not reach the boundary of harm: RR = 1·45 (1·06 to 1·99, p = 0·02). (Fig B in S6 Appendix). TSA for females with a 33% RRR was also inconclusive, despite a significant negative effect (Fig C in S6 Appendix). More details on the HTMV can be found in S6 Appendix.

Grading of recommendations, assessment, development, and evaluation & quality assessment

All articles were assessed for risk of bias using the ROB-2 tool (S2 Table). This evaluation was used in the completion of a GRADE assessment. GRADE assessments were made for each primary outcome in the protocol. Some trials investigated several outcomes and were included more than once in the GRADE table (S6-S7 Table).

The GRADE assessment for morbidity was low due to lack of blinding and the use of different interventions in the trials (S7 Table). For mortality, the lack of blinding of caregivers was not deemed a serious bias due to the indisputability of the outcome. Two GRADE assessments were made for mortality. One considered the overall body of evidence for mortality. Because this included both re-analyses, sub studies, and different intervention strategies, the overall quality rating was assessed to be low, reflecting the difficulty of drawing conclusions, despite the large amount of research in the field. However, during the review process, three large RCTs were identified and meta-analysed. These trials included a mortality outcome which reduced the impact of the lack of double blinding, had comparable interventions, and included 21,324 children in total. Thus, the GRADE assessment of these three RCTs alone was moderate to high (S7 Table). These trials were also applicable in a TSA. The z-curve for the TSA, based on these three trials, reached the area of futility, which leads to the conclusion of no non-specific effect on mortality after an extra early STMV dose.

Discussion

In this systematic review and meta-analysis, we investigated whether measles-containing vaccines have beneficial non-specific effects on mortality and morbidity beyond protection against measles. In accordance with Cochrane standards, only randomised trials were included. Gold standard meta-analysis methods were applied. The analysis found no support for beneficial non-specific effects of STMV. HTMV was linked to increased female mortality, but the result was not conclusive. Our findings align with a 2016 study by Higgins et al. [39] which found no such effects in randomised trials, but only in observational studies. This discrepancy was likely explained by healthy-and-wealthy-vaccinée bias [40,41] which is not present in randomised controlled trials [39].

Standard-titre measles vaccines

Eight original trials showed no significant associations between MCV and morbidity or mortality. TSA based on three large, comparable trials found that enough data was included to draw this conclusion. The RRR level was 25% in the TSA to align with RRRs investigated in the included RCT’s. Some upper confidence limits exceeded 1·25 in the forest plots, representing uncertainty in STMV effects. However, the TSA reached the area of futility, confirming that enough children were included to reject the hypothesised non-specific effects.

Three original RCT’s [11,12,16] also presented secondary analyses with follow-up restricted to 9 months. This shorter follow-period represented the time between the first interventional early MCV and the second routine MCV and enabled comparison between zero versus one dose of MCV. The three RCT’s were all large, with a combined population of 21,324 individuals, acceptable risks of bias, and highly comparable. None of the trials found a mortality difference between one and zero doses before nine months of age. The forest plot and TSA analysis showed no significant difference in mortality, and the area of futility was reached when RRR = 33% was investigated.

TSA showed that enough data was included to reject the hypothesis of non-specific effects in males (RRR = 25%) and in females (RRR = 33%).

For the outcome of morbidity, three RCTs [24,26,29], comprising one original and two re-analyses investigated a one vs zero dose intervention as a primary analysis. All RCTs were large with a combined population of 17,449 individuals. They had acceptable risks of bias and were highly comparable. None of these RCTs found a statistically significant difference in morbidity with a one vs zero dose intervention. The meta-analysis showed no significant difference in the forest plot and TSA, and this finding was conclusive in the TSA allowing us to conclude that there was no difference in morbidity between infants by sex, or by zero versus one dose of MCV.

High titre measles vaccines

HTMVs were discontinued in 1992 by the World Health Organization due to reports on/concern regarding negative effects on long term non-measles related mortality [42]. The negative effect was corroborated by the meta-analysis strategy used throughout this review (S6 Appendix). This is likely a specific effect of the higher dose of attenuated measles virus [43]. Hence, this finding does not support the hypothesis of non-specific vaccine effects.

Strengths and limitations

The strengths of the present systematic review were the gold standard meta-analytical approaches, including forest plots, and Trial Sequential Analysis. Only randomised controlled trials were included, and all analyses were pre-planned. Large populations were included in the meta-analyses, and an overview of all included trials and their re-analyses was created to ensure that meta-analyses did not include overlapping populations. The GRADE approach was used to assess the quality of evidence (S7 Table). Minor effects cannot be ruled out, but the RRR detection levels of the TSA were aligned with those of the included RCTs. Forest plots with both fixed and random effects were carried out for both crude and adjusted estimates. Only trials with a higher overall GRADE assessment were included in the TSA to minimise the heterogeneity and increase the quality of the results, which is a strength of this review. The statistical heterogenicity (I2) was below 10% in all main meta-analyses.

Limitations included the fact that some trials lost many participants during follow-up, potentially leading to attrition bias or dependent censoring. No other missing data was found during the review process. Further, trials were included in the overall meta-analysis irrespective of the assessed risk of bias. Many studies had a high risk of bias due to inadequate blinding necessitated by ethical considerations. Blinding was avoided to prevent caregivers from assuming their child had been vaccinated leading to missed vaccine doses. The limitation of lack of blinding was less serious for the indisputable outcome of mortality.

In some forest plots, wide confidence intervals were observed despite low heterogeneity estimates, potentially due to small sample sizes. Caution is warranted when interpreting p-values in analyses based on a small number of studies, as statistical power is reduced.

Another limitation was the utilisation of different interventions. As a trade-off between including all available RCT results and getting as close as possible to the true effect, different interventions were accepted in the forest plots. However, in the TSA, only fully comparable trials (with same intervention ‘two dose vs. one dose’ and ‘one dose vs. zero doses regimes’) were included. Neither the forest plots nor the TSAs revealed any sign of non-specific effects both if all available RCT’s were included (as in the forest plots) and if only fully comparable RCT’s were included (as in the TSAs).

Finally, due to the limited number of trials included, more formal assessments of publication bias (e.g., funnel plots and Egger’s test) were not feasible. Thus, we did not conduct publication bias analyses. However, we note that if publication bias were present, – meaning studies finding no evidence of non-specific effects were less likely to be published – it would strengthen the conclusion that such an effect is absent, as the available evidence would be biased/skewed toward showing an effect.

Conclusion

This systematic review and meta-analysis found no support for beneficial non-specific effects of STMV, but linked HTMV to increased female mortality.

Supporting information

S1 Appendix. Supplementary methods.

(DOCX)

pone.0321982.s001.docx (27.4KB, docx)
S1 Fig. Overview of trial populations.

Shows how the included trials were categorised by intervention, outcome and study populations.

(DOCX)

pone.0321982.s002.docx (91.2KB, docx)
S1 File. Citations excluded with animal and language filters.

(PDF)

pone.0321982.s003.pdf (690.6KB, pdf)
S1 Table. Systematic search string.

(DOCX)

pone.0321982.s004.docx (16.3KB, docx)
S2 Table. Risk of bias assessment.

(DOCX)

pone.0321982.s005.docx (18.1KB, docx)
S3 Table. Study characteristics and data included in main meta-analyses.

(DOCX)

pone.0321982.s006.docx (19.3KB, docx)
S4 Table. Full data extraction including estimates and 95% confidence intervals.

(DOCX)

pone.0321982.s007.docx (35.8KB, docx)
S5 Table. Exclusion reasons for all excluded studies after full text screening.

(DOCX)

pone.0321982.s008.docx (29.8KB, docx)
S6 Table. Decisions made in the TSA program in chronological order.

(DOCX)

pone.0321982.s009.docx (15.7KB, docx)
S2 Fig. Standard titre measles vaccine. Mortality. Two versus one doses. Crude data.

(DOCX)

pone.0321982.s010.docx (29KB, docx)
S2 Appendix. The standard titre measles vaccine. Mortality. One versus zero doses.

(DOCX)

pone.0321982.s011.docx (228.8KB, docx)
S3 Appendix. The standard titre measles vaccine. Mortality. Potential sex-differential effects.

(DOCX)

pone.0321982.s012.docx (543KB, docx)
S3 Fig. Morbidity. Standard titre measles vaccine. Two versus one doses. Crude data.

(DOCX)

pone.0321982.s013.docx (29.5KB, docx)
S4 Appendix. The standard titre measles vaccine. Morbidity. One versus zero doses.

(DOCX)

pone.0321982.s014.docx (162.2KB, docx)
S5 Appendix. The standard titre measles vaccine. Morbidity. Potential sex-differential effects.

(DOCX)

pone.0321982.s015.docx (217.9KB, docx)
S6 appendix. The high titre measles vaccine.

(DOCX)

pone.0321982.s016.docx (278KB, docx)
S7 Table. GRADE.

(DOCX)

pone.0321982.s017.docx (52.9KB, docx)

Acknowledgments

The lead author (the manuscript’s guarantor) affirms that the manuscript is an honest, accurate, and transparent account of the study being reported. No important aspects of the study have been omitted and any discrepancies from the study as planned have been explained. All authors attest they meet the ICMJE criteria for authorship.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

The author(s) received no specific funding for this work.

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PLoS One. 2025 Jul 2;20(7):e0321982. doi: 10.1371/journal.pone.0321982.r001

Author response to Decision Letter 0


Transfer Alert

This paper was transferred from another journal. As a result, its full editorial history (including decision letters, peer reviews and author responses) may not be present.

27 Jul 2024

Decision Letter 0

Ahmad Khalid Aalemi

Dear Dr. Fournais,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Additional Editor Comments :

Dear Louise,

Please revise your manuscript based on the reviewer comments.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Partly

Reviewer #4: Yes

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2. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

Reviewer #4: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

Reviewer #1: As written, the introduction does not provide an adequate argument for the need for the study. The background should be very strong to persuade the reader. Need more references for citing in the discussion. The language of the article will become even better and more understandable with the revision.

Reviewer #2: Review the information between lines 311 to 315, there is some extra spaces and the information could be adjusted to stay in the same paragraph.

The manuscript is easy to read and the information is clear. I would suggest make the figures bigger, except the figure 1, and colorful to make the differences or not more visible.

Reviewer #3: Overall Assessment

This manuscript presents a systematic review and meta-analysis assessing the hypothesis that standard-titre measles vaccines (STMV) have beneficial non-specific effects on mortality and morbidity. The study follows PRISMA guidelines and includes Trial Sequential Analysis (TSA) to ensure robustness. The authors conclude that their meta-analysis of randomized controlled trials (RCTs) does not support the hypothesis of non-specific effects of STMV, while historical high-titre measles vaccines (HTMV) were associated with increased female mortality.

The manuscript is well-structured, follows rigorous methodological standards, and provides a valuable contribution to the ongoing discussion about the non-specific effects of vaccines. However, there are areas where improvements could be made, particularly in the depth of discussion, clarity in statistical reporting, and consideration of potential limitations.

Major Strengths

1. Rigorous Methodology and Meta-Analysis Approach

• The study adheres to PRISMA guidelines and follows Cochrane Handbook recommendations for systematic reviews.

• The use of TSA is a significant strength, ensuring that conclusions are drawn from an adequate amount of data.

• Inclusion criteria were stringent, only considering RCTs, which minimizes bias from observational studies.

2. Comprehensive Literature Review

• The authors conducted a systematic search in major databases (EMBASE and PubMed) and repeated it weekly for nearly a year.

• The screening process involved independent reviewers with a clear conflict-resolution strategy.

3. Robust Statistical Analysis

• Meta-analysis includes both fixed and random effects models, which appropriately account for heterogeneity.

• TSA provides a higher level of confidence in the conclusions by assessing whether sufficient data exist to confirm or reject the hypothesis.

• Sex-stratified analyses enhance the understanding of differential effects.

Major Weaknesses and Areas for Improvement

1. Interpretation of Findings

• The authors state that their meta-analysis “did not support the hypothesis of non-specific effects of STMV.” However, the confidence intervals in several analyses are wide, and the rejection of a 25% or 33% risk reduction does not rule out smaller potential benefits.

• The authors could discuss the implications of their findings in the context of potential smaller effects that might be clinically relevant but undetectable within the current dataset.

2. Statistical Considerations

• The TSA was performed using a predefined 25% RRR, but this threshold is somewhat arbitrary. The authors should justify why this specific reduction was chosen.

• While TSA helps assess whether sufficient data have been collected, some meta-analyses still showed confidence intervals that include potential small effects. This should be discussed more explicitly.

3. Discussion on Potential Biases

• While RCTs are considered the highest level of evidence, there are limitations:

• Many trials had substantial loss to follow-up, which can introduce attrition bias.

• The lack of blinding in many trials could have influenced results, particularly for morbidity outcomes where subjective reporting may be involved.

• Heterogeneity among studies, particularly regarding intervention timing and population characteristics, could have affected outcomes. This heterogeneity should be explored more thoroughly in the discussion.

4. Consideration of Alternative Explanations

• Some prior observational studies have reported non-specific effects of measles vaccines, whereas this meta-analysis does not find such effects in RCTs. The authors should explore why observational studies might have produced different results (e.g., confounding, healthy vaccinee bias).

• The review does not consider potential age-related immune system differences that might influence vaccine effects. Could earlier vaccination (before 9 months) have an impact that was not captured in the TSA?

Minor Issues and Suggestions

1. Clarity in Presentation

• Some of the statistical results in the text could be more clearly presented. For example, rather than just stating “TSA showed that enough data was collected to conclusively reject the hypothesis,” the authors should specify the exact thresholds and explain them in a more intuitive manner.

• Figures and tables should be better integrated into the discussion. The manuscript refers to supplementary figures often, but some key findings should be highlighted directly in the main text.

2. Ethical Considerations

• The manuscript states that ethical approval was not needed. However, it would be useful to briefly explain why, especially since some of the included studies involved human participants.

3. Formatting and Writing Style

• The manuscript is generally well-written, but some sentences are long and difficult to follow. Simplifying complex statements and breaking them into smaller sentences would improve readability.

Conclusion and Recommendation

This manuscript presents a well-executed systematic review and meta-analysis that rigorously evaluates the hypothesis of non-specific effects of measles vaccines. While the findings do not support a strong beneficial effect of STMV, the discussion could be improved by acknowledging the potential for smaller effects, addressing study limitations in more depth, and better justifying statistical choices.

Reviewer #4: This is a clearly written systematic review and meta-analysis of randomised controlled trials with both standard titre and high titre live attenuated measles containing vaccines as intervention and other vaccines or placebo as comparator. The primary outcomes were mortality and morbidity Other secondary outcomes were examined as well. The analysis was interesting in that the authors added the Trial Sequential Analysis (TSA) approach having some advantageous features which was explained in the supplement.

The systematic review was routine and well done in that the investigators used PRISMA and PROSPERO. The studies included were only RCT’s . The approach to the Systematic search and screening and Quality assessment, resolution of reviewer differences as well as data extraction were explained adequately. Risk of bias assessment and other such features were reasonably outlined in the supplemental material. Obviously, publication bias was not discussed as the number of articles was perhaps inadequate for such. This should be made known to the reader.

Analysis was presented with Forest plots and the usual efficacy and heterogeneity type statistics were included.

The analysis appeared to confirm that the present review and meta-analysis did not support the hypothesized beneficial non-specific effects of STMV and found the historical HTMV to be possibly associated with increased female mortality.

There are some minor statistical concerns. The limitations are adequately noted by the investigators . However, they should mention to the reader, as noted above, why the lack of publication bias discussion, unless this reviewer missed it. Also, they should note the small number of articles in Figures S9 and S11 prompting one to interpret any p-values with caution. Also, any discussion would be helpful of heterogeneity or other causes of unusually wide confidence bands on any of the Forest plots both in the manuscript and supplement.

**********

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Reviewer #1: Yes:  DR. YAHYA H. Y. ALFARRA, BDS (Hons), MSc, PhD

Reviewer #2: No

Reviewer #3: Yes:  Mohamed Samy Abousenna

Reviewer #4: No

**********

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PLoS One. 2025 Jul 2;20(7):e0321982. doi: 10.1371/journal.pone.0321982.r003

Author response to Decision Letter 1


7 May 2025

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Author’s reply: Thank you. The manuscript has now been updated to fit the PLOS ONE style requirements and file naming.

2. As required by our policy on Data Availability, please ensure your manuscript or supplementary information includes the following:

A numbered table of all studies identified in the literature search, including those that were excluded from the analyses.

For every excluded study, the table should list the reason(s) for exclusion.

Author’s reply: Thank you. A new table (S5 Table) has now been added to the supplementary materials showing all exclusion reasons for our 69 excluded articles after full text screening.

If any of the included studies are unpublished, include a link (URL) to the primary source or detailed information about how the content can be accessed.

Author’s reply: No included studies are unpublished.

A table of all data extracted from the primary research sources for the systematic review and/or meta-analysis. The table must include the following information for each study:

Name of data extractors and date of data extraction

Author’s reply: Thank you. S4 Table has now been updated and includes name of data extractors and date.

Confirmation that the study was eligible to be included in the review.

Author’s reply: Thank you. S4 Table now includes a new column confirming whether each study was eligible for inclusion and whether each study was eligible for metaanalysis

All data extracted from each study for the reported systematic review and/or meta-analysis that would be needed to replicate your analyses.

Author’s reply: Thank you. All data needed to replicate our analyses can now be easily accessed in S4 Table.

If data or supporting information were obtained from another source (e.g. correspondence with the author of the original research article), please provide the source of data and dates on which the data/information were obtained by your research group.

Author’s reply: No data were from another source.

If applicable for your analysis, a table showing the completed risk of bias and quality/certainty assessments for each study or outcome. Please ensure this is provided for each domain or parameter assessed. For example, if you used the Cochrane risk-of-bias tool for randomized trials, provide answers to each of the signalling questions for each study. If you used GRADE to assess certainty of evidence, provide judgements about each of the quality of evidence factor. This should be provided for each outcome.

Author’s reply: A GRADE and a risk of bias assessment table has been included in supplementary materials. S2 Table and S7 Table.

An explanation of how missing data were handled.

This information can be included in the main text, supplementary information, or relevant data repository. Please note that providing these underlying data is a requirement for publication in this journal, and if these data are not provided your manuscript might be rejected.

Author’s reply: Thank you. Elaboration on missing data was included in the discussion l 321-323. PLEASE NOTE that all line numbers that are given in this document are based on looking at the manuscript with track changes. Track changes should be shown with ‘simple markup’ in word.

3. In the online submission form, you indicated that data sharing: Data and analyses will be available on request from the corresponding author (LAF).

All PLOS journals now require all data underlying the findings described in their manuscript to be freely available to other researchers, either 1. In a public repository, 2. Within the manuscript itself, or 3. Uploaded as supplementary information.

This policy applies to all data except where public deposition would breach compliance with the protocol approved by your research ethics board. If your data cannot be made publicly available for ethical or legal reasons (e.g., public availability would compromise patient privacy), please explain your reasons on resubmission and your exemption request will be escalated for approval.

Author’s reply: All data that were available on request will now be a part of supplementary material. See S1 file and S5 Table.

4. Please amend your list of authors on the manuscript to ensure that each author is linked to an affiliation. Authors’ affiliations should reflect the institution where the work was done (if authors moved subsequently, you can also list the new affiliation stating “current affiliation:….” as necessary).

Author’s reply: Thank you. Affiliations have been updated to include both the affiliation where the work of this paper was primarily carried out, and the current affiliation.

5. Please remove all personal information, ensure that the data shared are in accordance with participant consent, and re-upload a fully anonymized data set

Note: spreadsheet columns with personal information must be removed and not hidden as all hidden columns will appear in the published file.

Additional guidance on preparing raw data for publication can be found in our Data Policy (https://journals.plos.org/plosone/s/data-availability#loc-human-research-participant-data-and-other-sensitive-data) and in the following article: http://www.bmj.com/content/340/bmj.c181.long.

Author’s reply: Thank you. No personal information was included in the manuscript. No participant consent was obtained before the beginning of this paper since all data from this paper was already published in other papers that had the responsibility to obtain participant consent. This reasoning has now been added to the manuscript p. 15 l. 366-367

6. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

Author’s reply: Thank you. All supporting information files are now captioned at the end of the manuscript and in the correct chronological order. Furthermore in-text citations match. The supplementary files will be uploaded according to the provided guidelines.

Additional Editor Comments :

Dear Louise,

Please revise your manuscript based on the reviewer comments.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Partly

Reviewer #4: Yes

________________________________________

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

Reviewer #4: Yes

________________________________________

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

________________________________________

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

________________________________________

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: As written, the introduction does not provide an adequate argument for the need for the study. The background should be very strong to persuade the reader. Need more references for citing in the discussion. The language of the article will become even better and more understandable with the revision.

Author’s reply: Thank you for this valuable feedback. In the revised manuscript, the Background and Discussion sections have been elaborated upon accordingly.

Reviewer #2: Review the information between lines 311 to 315, there is some extra spaces and the information could be adjusted to stay in the same paragraph.

Author’s reply: Thank you. The text was revised accordingly.

The manuscript is easy to read and the information is clear. I would suggest make the figures bigger, except the figure 1, and colorful to make the differences or not more visible.

Author’s reply: Thank you. All supplementary figures have been made bigger and/or more colourful. Colors have been added to all figures in the manuscript. But the figures in the main text must comply with the requirements from PLOS ONE and therefore this will dictate the size of these figures.

Reviewer #3: Overall Assessment

This manuscript presents a systematic review and meta-analysis assessing the hypothesis that standard-titre measles vaccines (STMV) have beneficial non-specific effects on mortality and morbidity. The study follows PRISMA guidelines and includes Trial Sequential Analysis (TSA) to ensure robustness. The authors conclude that their meta-analysis of randomized controlled trials (RCTs) does not support the hypothesis of non-specific effects of STMV, while historical high-titre measles vaccines (HTMV) were associated with increased female mortality.

The manuscript is well-structured, follows rigorous methodological standards, and provides a valuable contribution to the ongoing discussion about the non-specific effects of vaccines. However, there are areas where improvements could be made, particularly in the depth of discussion, clarity in statistical reporting, and consideration of potential limitations.

Major Strengths

1. Rigorous Methodology and Meta-Analysis Approach

• The study adheres to PRISMA guidelines and follows Cochrane Handbook recommendations for systematic reviews.

• The use of TSA is a significant strength, ensuring that conclusions are drawn from an adequate amount of data.

• Inclusion criteria were stringent, only considering RCTs, which minimizes bias from observational studies.

2. Comprehensive Literature Review

• The authors conducted a systematic search in major databases (EMBASE and PubMed) and repeated it weekly for nearly a year.

• The screening process involved independent reviewers with a clear conflict-resolution strategy.

3. Robust Statistical Analysis

• Meta-analysis includes both fixed and random effects models, which appropriately account for heterogeneity.

• TSA provides a higher level of confidence in the conclusions by assessing whether sufficient data exist to confirm or reject the hypothesis.

• Sex-stratified analyses enhance the understanding of differential effects.

Major Weaknesses and Areas for Improvement

1. Interpretation of Findings

• The authors state that their meta-analysis “did not support the hypothesis of non-specific effects of STMV.” However, the confidence intervals in several analyses are wide, and the rejection of a 25% or 33% risk reduction does not rule out smaller potential benefits.

• The authors could discuss the implications of their findings in the context of potential smaller effects that might be clinically relevant but undetectable within the current dataset.

Author’s reply: Thank you. In the Discussion of the revised manuscript, this aspect was clarified. l. 314-317.

2. Statistical Considerations

• The TSA was performed using a predefined 25% RRR, but this threshold is somewhat arbitrary. The authors should justify why this specific reduction was chosen.

Author’s reply: Thank you. As also mentioned in the manuscript, the RRRs of the TSAs were consequently aligned with those of the included RCTs. In the Discussion of the revised manuscript, this aspect was elaborated upon. l. 278

• While TSA helps assess whether sufficient data have been collected, some meta-analyses still showed confidence intervals that include potential small effects. This should be discussed more explicitly.

Author’s reply: Thank you. In the Discussion of the revised manuscript, this aspect was clarified. l. 329-331

3. Discussion on Potential Biases

• While RCTs are considered the highest level of evidence, there are limitations:

• Many trials had substantial loss to follow-up, which can introduce attrition bias.

Author’s reply: Thank you. In the Limitations section of the Discussion of the revised manuscript, attrition bias was elaborated upon lines 321-323.

• The lack of blinding in many trials could have influenced results, particularly for morbidity outcomes where subjective reporting may be involved.

Author’s reply: Thank you. In the Limitations section of the Discussion of the revised manuscript, lack of blinding was elaborated upon lines 323-327.

• Heterogeneity among studies, particularly regarding intervention timing and population characteristics, could have affected outcomes. This heterogeneity should be explored more thoroughly in the discussion.

Author’s reply: Thank you. In the Limitations section of the Discussion of the revised manuscript, the important aspect of heterogeneity, which was measured to be below 10% throughout the present systematic review, was elaborated upon lines 319-320.

4. Consideration of Alternative Explanations

• Some prior observational studies have reported non-specific effects of measles vaccines, whereas this meta-analysis does not find such effects in RCTs. The authors should explore why observational studies might have produced different results (e.g., confounding, healthy vaccinee bias).

Author’s reply: Thank you. The importance of confounding and healthy-and-wealthy-vaccinée bias (inclusive relevant references) have been added to the Discussion of the revised manuscript. Lines 270-273

• The review does not consider potential age-related immune system differences that might influence vaccine effects. Could earlier vaccination (before 9 months) have an impact that was not captured in the TSA?

Author’s reply: Thank you for this relevant question, which was addressed in the 0 vs. 1 dose of MCV analysis described separately in the results section. Also for early MCV administration, no non-specific vaccine effects were observed.

Minor Issues and Suggestions

1. Clarity in Presentation

• Some of the statistical results in the text could be more clearly presented. For example, rather than just stating “TSA showed that enough data was collected to conclusively reject the hypothesis,” the authors should specify the exact thresholds and explain them in a more intuitive manner.

Author’s reply: Thank you. A more intuitive explanation of thresholds and impact of the figures has been added to the manuscript when the first TSA fig 2 is presented. Lines 153-159.

• Figures and tables should be better integrated into the discussion. The manuscript refers to

Attachment

Submitted filename: Response to reviewers.docx

pone.0321982.s018.docx (42.4KB, docx)

Decision Letter 1

Ahmad Khalid Aalemi

Measles vaccines and non-specific effects on mortality or morbidity. A systematic review and meta-analysis.

PONE-D-24-29586R1

Dear Dr. Louis Amstrup Fournis,

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Reviewer's Responses to Questions

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Reviewer #2: All comments have been addressed

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Reviewer #2: (No Response)

Reviewer #3: Thank you for your thoughtful and thorough revision of the manuscript titled "Measles vaccines and nonspecific effects on mortality or morbidity. A systematic review and meta-analysis." I appreciate the effort you made to address each of the points raised during the initial review.

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Acceptance letter

Ahmad Khalid Aalemi

PONE-D-24-29586R1

PLOS ONE

Dear Dr. Fournais,

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Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Appendix. Supplementary methods.

    (DOCX)

    pone.0321982.s001.docx (27.4KB, docx)
    S1 Fig. Overview of trial populations.

    Shows how the included trials were categorised by intervention, outcome and study populations.

    (DOCX)

    pone.0321982.s002.docx (91.2KB, docx)
    S1 File. Citations excluded with animal and language filters.

    (PDF)

    pone.0321982.s003.pdf (690.6KB, pdf)
    S1 Table. Systematic search string.

    (DOCX)

    pone.0321982.s004.docx (16.3KB, docx)
    S2 Table. Risk of bias assessment.

    (DOCX)

    pone.0321982.s005.docx (18.1KB, docx)
    S3 Table. Study characteristics and data included in main meta-analyses.

    (DOCX)

    pone.0321982.s006.docx (19.3KB, docx)
    S4 Table. Full data extraction including estimates and 95% confidence intervals.

    (DOCX)

    pone.0321982.s007.docx (35.8KB, docx)
    S5 Table. Exclusion reasons for all excluded studies after full text screening.

    (DOCX)

    pone.0321982.s008.docx (29.8KB, docx)
    S6 Table. Decisions made in the TSA program in chronological order.

    (DOCX)

    pone.0321982.s009.docx (15.7KB, docx)
    S2 Fig. Standard titre measles vaccine. Mortality. Two versus one doses. Crude data.

    (DOCX)

    pone.0321982.s010.docx (29KB, docx)
    S2 Appendix. The standard titre measles vaccine. Mortality. One versus zero doses.

    (DOCX)

    pone.0321982.s011.docx (228.8KB, docx)
    S3 Appendix. The standard titre measles vaccine. Mortality. Potential sex-differential effects.

    (DOCX)

    pone.0321982.s012.docx (543KB, docx)
    S3 Fig. Morbidity. Standard titre measles vaccine. Two versus one doses. Crude data.

    (DOCX)

    pone.0321982.s013.docx (29.5KB, docx)
    S4 Appendix. The standard titre measles vaccine. Morbidity. One versus zero doses.

    (DOCX)

    pone.0321982.s014.docx (162.2KB, docx)
    S5 Appendix. The standard titre measles vaccine. Morbidity. Potential sex-differential effects.

    (DOCX)

    pone.0321982.s015.docx (217.9KB, docx)
    S6 appendix. The high titre measles vaccine.

    (DOCX)

    pone.0321982.s016.docx (278KB, docx)
    S7 Table. GRADE.

    (DOCX)

    pone.0321982.s017.docx (52.9KB, docx)
    Attachment

    Submitted filename: Response to reviewers.docx

    pone.0321982.s018.docx (42.4KB, docx)

    Data Availability Statement

    All relevant data are within the paper and its Supporting Information files.


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