Gene Signatures of Influenza Vaccine Responses in Older Adults: Complete Statistical Analysis

1. Trial at a Glance

This page covers the registry results for Gene Signatures of Influenza Vaccine Responses in Older Adults (NCT03603509), a randomized phase 4 study. The primary outcome is Innate Cell IFNa2a Production.

2. Clinical Question

The study concerns immune responses to influenza vaccination in people aged 65 years and older. Its broad research aim is to understand responses to an adjuvanted subunit vaccine and a high-dose vaccine, and how immune response changes with age. The primary endpoint listed in the registry record is not a clinical illness outcome: it is interferon alpha 2a production after blood samples were stimulated with influenza virus in vitro.

The question is therefore about a laboratory measure of immune response, not directly about prevention of influenza, severity of illness, or other patient outcomes. Those outcomes are not reported in the registry record as primary results. The study also lists secondary measures involving T-cell responses and phenotypes, innate-cell phenotype, cytomegalovirus serostatus, and the CD4/CD8 ratio. The record does not give the numerical results for those measures in the information summarized in this analysis.

3. Trial Design

The registry record describes a Phase 4, randomized, unmasked, parallel-group study sponsored by Mayo Clinic. Enrollment was 241. The study summary describes participants as people aged 65 years and older and states that the research examined responses to Fluad and Fluzone High-Dose. The arm descriptions specify a single dose of the assigned vaccine. The record lists 120 participants at risk for adverse-event reporting in the Fluad group and 121 in the Fluzone group.

The study started on 2018-08-27, reached primary completion on 2019-01-02, and is listed as completed. These dates describe the registry record; they do not establish when every laboratory measurement or analysis was performed. The registry record does not report further details about the randomization procedure, allocation concealment, recruitment, eligibility criteria beyond the age group stated in the summary, or how participants and investigators were kept unmasked.

The primary outcome is innate-cell IFNa2a production, measured in pg/mL as a median with an inter-quartile range. Its stated timeframe is baseline and Day 1. The assay description says cytokine secretion was measured after in-vitro stimulation with influenza virus. This is a laboratory endpoint, rather than a clinical endpoint such as influenza infection. The record notes that the analyzed sample count was lower than the study cohort total because some serum samples failed assay quality control, were degraded, or failed to function properly in the assay.

The registry lists 12 outcome measures and 2 analyses. It also indicates that the primary outcome has several categories or time points and was not reduced to one comparison. The details of those categories, the analysis methods, and the results for the primary endpoint are not reported in the registry record. No sample-size justification or planned interim analysis is reported in the registry record. Any planning figures elsewhere should be read as illustrations based on an observed effect and stated assumptions, not as the trial's original design rationale or as evidence of observed power.

4. Participant Flow and Accounting

The registry reports how participants moved through each stage of the study. All counts below are exactly as posted; missing counts are not inferred from other stages.

Overall Study

Reasons for not completing this period:

Analysis population for the primary outcome, as defined in the registry: The number of samples analyzed is less than the study cohort total because some of the serum samples failed in the assay. These failures were either: results did not meet QC, sample degraded and failed to function in the assay properly.

5. Baseline Characteristics

The table reproduces the baseline characteristics posted in the registry, which describe the participants analysed at baseline and help show whether the randomized groups were comparable.

6. Primary Endpoint

The registry lists the primary outcome as Innate Cell IFNa2a Production, measured over Baseline, Day 1.

The primary endpoint is innate-cell production of interferon alpha 2a (IFNa2a), reported in pg/mL. The registry describes the measurement as cytokine secretion after serum samples were stimulated with influenza virus in vitro. Its listed time points are baseline and Day 1, and the planned summary form is a median with an inter-quartile range.

As a general statistical explanation, a laboratory response endpoint can describe a biological process under controlled assay conditions. It can help characterize how an immune system responds to a stimulus without requiring that a participant first experience influenza illness. That makes it distinct from a clinical endpoint such as infection, complications, or symptoms. The registry record does not report those clinical outcomes as the primary endpoint.

The median describes the central point of a distribution when observations are ordered. The inter-quartile range describes the span of the middle portion of that distribution. These summaries are often useful when measurements are skewed or include extreme values, because they are less affected by unusually high or low observations than the mean and standard deviation. This is a general explanation of the summary statistics; the registry record does not report the observed medians or inter-quartile ranges for the endpoint.

The record also cautions that fewer samples were analyzed than the study cohort total. Some serum samples did not pass quality control, were degraded, or did not function properly in the assay. The numbers of failed samples and the resulting analysis counts are not reported in the registry record. This matters because the endpoint summarizes usable laboratory measurements, not necessarily every randomized participant. The registry record does not describe how missing assay measurements were handled in the endpoint analysis.

7. Why No Re-analysis Is Shown

8. Statistical Interpretation

9. Statistical Methods Explained

The registry record identifies a randomized, parallel-group design without masking. In a randomized study, assignment is intended to create groups that can be compared while limiting systematic differences at the point of assignment. This is a general description of randomization, not a claim about the specific sequence-generation or concealment procedures used in this trial; those procedures are not reported in the registry record. The record's unmasked designation means the study is listed as having no masking. It does not specify which study roles knew the assigned vaccine.

What does the endpoint summary represent?

The primary outcome is IFNa2a production in pg/mL after in-vitro stimulation with influenza virus, summarized using a median and an inter-quartile range. In general, a median locates the middle of ordered measurements, while the inter-quartile range describes the spread of the central portion of those measurements. These summaries can be useful for biological measurements whose distributions are asymmetric. The registry record does not provide the actual summaries, so no observed central value or spread can be described.

Baseline and Day 1 are the listed time points. A baseline measurement can provide a reference before or around the intervention, while a later measurement can describe a response at a subsequent time. That is a general interpretation of repeated measurements, not a claim about the exact sampling schedule or analysis used in this trial. The registry record does not report the exact timing, whether the groups were compared at each time point, or whether a within-person change was analyzed.

How should uncertainty be understood?

General statistical interpretation distinguishes the size of an estimated effect from its uncertainty. An estimate describes a contrast or association in the observed data; an uncertainty interval indicates the range of values compatible with the data under the analysis assumptions. Confidence intervals are one familiar form of uncertainty interval. A p-value answers a narrower question about how compatible the data are with a specified null model. The registry record does not report a numerical primary-effect estimate, a confidence interval, or a p-value in the information summarized for this analysis. Without those results, neither statistical precision nor the strength of evidence for a group difference can be assessed.

Even when an estimate and interval are available, interpretation depends on the analysis specification: which participants are included, how repeated observations are handled, whether baseline values are adjusted for, and what contrast is being estimated. These details are not reported in the registry record. A laboratory endpoint also has measurement considerations, including assay reliability and quality control. The record specifically notes quality-control failures, degradation, and samples that did not function properly, but does not report their counts or the analysis decisions applied to them.

Why can over-dispersion matter?

General explanation: over-dispersion occurs when observed variability is greater than a statistical model expects. A model that understates variability can produce uncertainty estimates that are too narrow and tests that appear more decisive than the data warrant. The relevance of this issue depends on the endpoint and model. The primary endpoint is described with medians and inter-quartile ranges, but the registry record does not report the model or inferential method used. It would therefore be inappropriate to assert that over-dispersion was present or that a particular correction was needed.

What can planning illustrations tell readers?

General explanation: sample-size calculations explore how many participants might be needed to detect an effect under chosen assumptions, such as a significance level, allocation pattern, and target power. When calculations are based on an observed effect, they are planning illustrations, not observed power and not proof that a study was adequately powered. The stated illustration assumptions for this study include a two-sided significance level of 0.05, equal allocation between two arms, and a normal approximation; the target effect is expressed as a fraction of the observed effect. The record's note also distinguishes participants per arm for non-time-to-event outcomes from total events for time-to-event outcomes. These assumptions describe the illustration, not the trial's own design justification. The trial's sample-size justification is not reported in the registry record.

Because the primary endpoint has several categories or time points and was not reduced to one comparison, a single sample-size interpretation may not correspond to every possible contrast. The registry record does not report which specific endpoint contrast underlies any planning illustration or how the observed effect was chosen. Such illustrations should therefore be read as conditional calculations rather than as estimates of the study's achieved evidential strength.

How should multiple outcomes be considered?

The registry lists 12 outcome measures and 2 analyses. In general, when multiple outcomes, time points, or comparisons are examined, the chance of at least one apparently unusual result can increase if each is tested separately without adjustment. Methods that account for multiplicity may change the interpretation of p-values or confidence intervals. The registry record does not report a multiplicity strategy, identify which results were designated confirmatory, or specify how the listed analyses map to the outcome measures. The primary endpoint's multiple categories or time points make the definition of the target comparison especially important.

The record lists secondary outcomes involving T-cell ELISPOT response, T-cell phenotype, innate-cell phenotype, CMV serostatus, and CD4/CD8 ratio. These may address different biological questions, but the numerical findings and their analysis details are not reported in the registry record. The endpoint list alone does not establish which analyses were prespecified, how missing measurements were handled, or whether any results were adjusted for multiple testing.

10. Multiplicity, Missing Data and Analysis Populations

The registry record lists 12 outcome measures and 2 analyses, and it indicates that the primary outcome includes several categories or time points rather than one reduced comparison. This means the target of inference cannot be represented by one unambiguous contrast from the registry description alone. The registry record does not report a multiplicity strategy, such as a procedure for controlling the interpretation of multiple tests, nor does it detail which analyses were primary, secondary, or exploratory.

As a general statistical point, examining multiple outcomes, time points, or group contrasts can increase the chance of finding an apparently notable result by chance when tests are treated independently. Multiplicity methods can help align the interpretation of uncertainty with the number and structure of analyses. The record does not report whether such methods were used, so no adjustment should be assumed. The two listed analyses do not by themselves reveal the number of tests or the analysis hierarchy.

Missing assay data are directly relevant to the primary endpoint. The registry states that fewer serum samples were analyzed than the study cohort total because some samples did not meet quality control, were degraded, or failed to function in the assay. The number of affected samples, counts by randomized group, and reasons by group are not reported in the registry record. The approach to handling these missing measurements is also not reported in the registry record.

The record does not state whether the primary endpoint analysis included all randomized participants, only participants with usable assay samples, or another defined analysis population. It also does not describe imputation, sensitivity analyses, or other methods for assessing the consequences of missing assay results. As a result, readers cannot determine from the registry record how much the endpoint analysis population differed from the enrolled cohort or whether missingness could have influenced the comparison.

11. Safety Results

The registry reports serious adverse events as affected participants divided by participants at risk.

Deaths

Other (non-serious) adverse events

Participants with at least one non-serious event at or above the registry's 0% reporting threshold.

Most frequent non-serious adverse event terms

The registry lists serious adverse events for 120 participants at risk in the Fluad group and 121 at risk in the Fluzone group. The number affected by serious adverse events is 0 in each group. Deaths are also listed as 0 in each group. The registry record lists no serious adverse-event terms.

For other adverse events, the registry lists 31 affected in the Fluad group and 22 in the Fluzone group, with 120 and 121 participants at risk, respectively. These are the posted counts; the registry information does not specify event types, severity, timing, relationship to vaccination, or whether counts represent distinct participants or event episodes. Those details are not reported in the registry record.

As a general statistical explanation, an at-risk count gives the group denominator associated with a safety category, while an affected count reports the number recorded in that category. A count alone does not describe the duration of observation or uncertainty around the underlying event frequency. No formal between-group safety comparison or uncertainty interval is reported in the registry record. These figures should be read as registry-reported safety counts, not as a complete assessment of all possible adverse events or as evidence about the primary immune-response endpoint.

12. What the Result Does, and Does Not, Mean

The registry record describes a randomized comparison of two influenza vaccines in adults aged 65 years and older, with a laboratory immune-response endpoint assessed at baseline and Day 1. This defines the scientific question and the kind of measurement used. It does not, by itself, establish the direction or size of any difference between groups. The numerical IFNa2a medians, inter-quartile ranges, and a single reduced comparison are not reported in the registry record.

The primary endpoint measures cytokine production after an in-vitro stimulus. As a general distinction, a laboratory response can inform understanding of immune activity, but it is not interchangeable with a clinical outcome such as influenza infection or illness. The registry record does not report a primary clinical efficacy outcome in the information summarized for this analysis. Therefore, no conclusion about prevention of illness or other clinical benefit follows from the endpoint description alone.

The study summary describes a broader goal of understanding how immune response diminishes with age in men and women and how influenza immunity develops after vaccination. The enrolled population is described as adults aged 65 years and older, but the registry information does not report a numerical comparison by age within that group or by sex. The broad motivation should not be mistaken for evidence that a specific age-related or sex-related pattern was demonstrated.

The record notes that assay failures reduced the number of samples analyzed relative to the cohort total. Because counts and missing-data methods are not reported in the registry record, the extent to which missing measurements could affect the endpoint estimate is unclear. The safety counts provide separate descriptive information, but they do not resolve the missing primary-endpoint results or establish a treatment comparison with quantified precision.

In short, the registry record establishes the design, intervention groups, endpoint definition, assay limitation, and selected safety counts. It does not report enough numerical primary-outcome information to judge the size, direction, or precision of the immune-response contrast. The study's stated aims and the existence of a completed trial do not substitute for those results.

13. Why This Trial Matters Statistically

This trial is statistically interesting because it combines a randomized parallel-group comparison with a laboratory endpoint measured at more than one time point. Randomization gives the design a framework for comparing assigned groups, while the baseline and Day 1 measurements raise questions about how time and within-participant observations were incorporated. The registry record identifies the design and time points, but does not report the detailed analysis needed to answer those questions.

The endpoint is summarized using medians and inter-quartile ranges rather than a mean and standard deviation. As a general statistical point, this can be a useful way to describe biological measurements when their distributions are asymmetric or contain extreme values. It also means that readers need the actual distribution summaries and a clearly defined group contrast to interpret the result. Those numerical primary-endpoint results are not reported in the registry record.

Assay reliability and missingness are particularly relevant in studies using laboratory outcomes. The record explicitly notes failed quality control, degradation, and samples that did not function properly. These are not merely abstract statistical concerns: they determine which measurements can contribute to the endpoint analysis. Since the number and distribution of failed samples and the handling approach are not reported in the registry record, the consequences for precision and potential selection effects cannot be evaluated.

The listed multiple outcomes and time points also make the analysis plan important. In general, a prespecified outcome hierarchy and multiplicity strategy help readers distinguish confirmatory questions from exploratory ones. The registry record lists the number of outcomes and analyses but does not describe a multiplicity approach or reduce the primary endpoint to one comparison. This limits the extent to which any result could be interpreted as a single definitive test even if individual summaries were available.

Finally, the record illustrates why study completion and enrollment totals are not enough to interpret a biological comparison. Readers need endpoint estimates, uncertainty, analysis populations, and analysis methods to assess what the data support. The registry record supplies useful design and measurement context, but those essential numerical and methodological details are not reported in the information.

14. Limitations

• The posted summary statistics are the only data these results rest on; patient-level data, full protocols and statistical analysis plans are not available.
• Recalculated results are simple, unadjusted calculations from posted summaries. They do not replace the sponsor's prespecified analysis.

A key limitation of the available registry information is that it does not report numerical primary-endpoint results. The endpoint is defined as IFNa2a production in pg/mL, summarized by median and inter-quartile range at baseline and Day 1, but the actual summaries and any group contrast are not reported in the registry record. The record also says the primary outcome has several categories or time points and was not reduced to one comparison, leaving the target contrast unclear.

The primary endpoint has assay-related missingness. The record attributes the lower analyzed sample count to samples that did not meet quality control, were degraded, or failed to function properly. It does not report the number of failures, their distribution between groups, or how the analysis handled missing measurements. The analysis population and any sensitivity assessment are not reported in the registry record.

Further details needed to assess the analysis are absent from the registry information. The statistical model, baseline adjustment, treatment of repeated measurements, uncertainty estimates, and any multiplicity procedure are not reported in the registry record. The record also does not report the trial's sample-size justification, randomization implementation, or whether any interim analyses occurred. These details should not be inferred from the study's randomized designation or from planning illustrations.

The safety information is limited to posted counts. Event definitions, type, severity, timing, attribution, and follow-up duration are not reported in the registry record. The secondary outcomes are named, but their numerical findings and analysis details are not included in the information. These limitations constrain interpretation of both the immune-response comparison and the broader study aims.

15. Related Tutorials

16. Related Statistical Calculators

17. Sources

• ClinicalTrials.gov: Gene Signatures of Influenza Vaccine Responses in Older Adults — NCT03603509.
• Registry record last updated 2023-05-16; status last verified 2023-04.
• Results first posted 2023-05-16.
• Data retrieved from ClinicalTrials.gov on 2026-11-09.
• Group sizes, means, event counts and sponsor-reported p-values are reproduced as posted. Effect estimates, confidence intervals and p-values not attributed to the sponsor are independently calculated from the posted summary statistics.

18. Record Summary

The registry record describes NCT03603509, “Gene Signatures of Influenza Vaccine Responses in Older Adults,” as a completed Phase 4 randomized, unmasked, parallel-group study sponsored by Mayo Clinic. Enrollment was 241. The study compared a single dose of Fluad with a single dose of Fluzone High-Dose in adults aged 65 years and older. The record lists a start date of 2018-08-27 and primary completion date of 2019-01-02.

The primary endpoint is innate-cell IFNa2a production in pg/mL after in-vitro stimulation with influenza virus, summarized as a median and inter-quartile range at baseline and Day 1. Some samples failed assay quality control, were degraded, or did not function, so fewer samples were analyzed than the study cohort total. Numerical primary-endpoint results and the handling of missing assay data are not reported in the registry record.

The registry lists 12 outcome measures and 2 analyses, and notes that the primary endpoint was not reduced to one comparison. Safety reporting lists no serious adverse events or deaths in either group, with other adverse events recorded in both groups. Further analysis and safety details are not reported in the registry record.