Immune Response and General Immune Health in Subjects Infected With Herpes Simplex Virus Type 1 (HSV-1): Complete Statistical Analysis

1. Trial at a Glance

This page covers the registry results for Immune Response and General Immune Health in Subjects Infected With Herpes Simplex Virus Type 1 (HSV-1) (NCT03661541), a non-randomized phase 1 study. The primary outcome is Ratio of Absolute Gene Expression of IFNG and IL5 in PBMCs Between 8 Weeks and Baseline.

2. Clinical Question

The study is framed around immune response and general immune health among people already infected with HSV-1. Blood samples were used to assess PBMC proliferation and immune-related gene expression after exposure in vitro to HSV-1 virus, HSV-1-infected cell extracts, or Candida albicans extract. Serum anti-HSV-1 IgG levels were also measured. The Candida extract is described as a test of general immune response.

The frequent-outbreak group was assessed at baseline and returned for blood collection on days 15 and 57 after the SADBE application. The summary describes the other groups as having baseline blood collection; the primary outcome timeframe identifies their measurements on day 1 in relation to the frequent-outbreak group. Because outbreak history determined group membership and treatment was not randomized, comparisons across groups are observational. The study can describe measured immune responses in these groups, but the registry record does not establish that between-group differences would be caused by SADBE or by outbreak frequency.

3. Trial Design

This is a phase I, parallel-group study with non-randomized allocation and no masking. The registry lists an enrollment of 36 and describes three groups based on self-reported herpes labialis outbreaks during the previous 12 months: six or more, one or two, and zero. The brief summary says twelve subjects were recruited in each group. Participants were HSV-1 antibody positive according to the study description.

The groups did not represent randomized treatment arms. The frequent-outbreak group received one topical application of 2% SADBE in DMSO after its baseline blood collection. It returned on days 15 and 57 for further blood collection. PBMCs were tested in vitro for proliferation and gene expression, and serum anti-HSV-1 IgG was measured. The registry describes the one-or-two-outbreak and zero-outbreak groups as matched to the frequent-outbreak group on demographic characteristics, but the matching details are not reported in the registry record.

The primary outcome description identifies gene expression for IFNG and IL5, measured by qRT-PCR crossing threshold (Ct), and names a comparison involving the frequent-outbreak group at day 57 and the other groups at day 1 relative to baseline for the frequent-outbreak group. The stated timeframe also refers to baseline and eight weeks after baseline, with a seven-day allowance for participants with fewer than six outbreaks. The relation between these descriptions is not further explained in the registry record.

The record lists the study as completed, with a start date of 2017-03-02 and primary completion on 2017-06-17. Those dates and completion status do not establish that outcome results were analyzed or reported. The registry lists three outcome measures but zero analyses. Statistical methods for the outcome analyses, including any adjusted comparisons, are not reported in the registry record.

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

Analysis population for the primary outcome, as defined in the registry: Absolute gene expression for Group A on Day 57, Group B on day 1 and Group C on day 1 compared to Group A on Day 1.

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 Ratio of Absolute Gene Expression of IFNG and IL5 in PBMCs Between 8 Weeks and Baseline, measured over Baseline and 8 weeks after baseline (Day 1) +/- 7 days for subjects with less than 6 herpres labialis outbreaks in 12 months, relative to baseline for subjects with 6 or greater herpes labialis outbreaks.

The primary endpoint is titled as a ratio of absolute gene expression of IFNG and IL5 in PBMCs between eight weeks and baseline. The measurement is qRT-PCR crossing threshold cycle (Ct). The registry description says RNA was assessed after PBMCs were exposed in vitro to four conditions: no stimulus, HSV-1 virus, HSV-1-infected cell extracts, and Candida albicans extract. It specifies that a lower Ct indicates higher gene expression, with a lower Ct of 1 described as approximately a 2-fold higher expression level.

In general, gene-expression measures can help characterize how cells respond to different stimuli. Assessing both IFNG and IL5 gives information about two immune-related genes; testing more than one stimulus allows responses to be considered in different laboratory conditions. These measurements are laboratory markers, not direct measures of clinical symptoms or outbreak prevention.

The endpoint title, timeframe, and population description use somewhat different comparison language. The population description identifies the frequent-outbreak group on day 57 and the other groups on day 1 relative to the frequent-outbreak group at day 1. The timeframe refers to eight weeks after baseline for the groups with fewer outbreaks, relative to baseline for the frequent-outbreak group. The registry record does not clarify this wording or report the endpoint values, the ratio estimates, or their uncertainty. It therefore does not show how gene expression changed or how precisely any change was estimated.

7. Why No Re-analysis Is Shown

8. Statistical Interpretation

9. Statistical Methods Explained

What does the Ct measure mean?

The endpoint uses the qRT-PCR crossing threshold cycle, or Ct, to quantify gene expression. In general, qRT-PCR detects genetic material through repeated amplification, and the threshold cycle records when the signal reaches a defined level. The registry describes lower Ct as corresponding to higher gene expression, and states that a Ct difference of 1 is approximately a 2-fold expression difference. This is the registry's interpretation of the measure; it does not supply observed Ct values or an analyzed contrast.

The endpoint names IFNG and IL5 and refers to absolute gene expression. It also describes PBMC exposure to no stimulus, HSV-1 virus, HSV-1-infected cell extracts, and Candida albicans extract. Each laboratory condition addresses a different stimulus context. In general, measurements from these conditions should be interpreted with attention to which stimulus and gene are being compared. The registry does not report the results for any condition.

What does a ratio of expression represent?

In general, a ratio expresses one quantity relative to another. For a before-and-after expression comparison, a ratio can describe how the later measurement relates to the baseline measurement. Its interpretation depends on the precise scale and calculation used. Here, the endpoint title refers to a ratio of absolute gene expression, while the assay description emphasizes Ct values, whose direction is inverse to expression. The exact calculation linking Ct to the stated ratio is not reported in the registry record. The estimate should therefore not be reconstructed from the endpoint wording.

How should group comparisons be read?

In general, a randomized comparison aims to make treatment groups comparable on average at the outset. This study is listed as non-randomized, and membership in the three groups followed outbreak history. The frequent-outbreak group received SADBE, whereas the other groups are described by their outbreak history and as matched to the frequent-outbreak group on demographic characteristics. Observed group differences could therefore reflect more than treatment. Matching may address specified characteristics, but the matching procedure, variables, and analysis are not reported in the registry record.

A within-group comparison over time is different from a comparison between groups. In general, a before-and-after contrast describes change in the measured group, but by itself cannot separate an intervention effect from time-related change or other influences. A between-group contrast can provide context, but in this design group membership and intervention exposure are not independent. The registry does not report an analysis that addresses these issues.

What do mean and standard deviation describe?

The primary outcome is identified as a continuous measure with a mean parameter and standard deviation as the dispersion measure. In general, a mean describes the average observed value, while the standard deviation describes the spread of individual values around that average. The standard deviation is not the same as the uncertainty in the estimated mean. Estimating uncertainty in a mean generally also requires information about the number of observations contributing to it and the analysis method. No outcome means, standard deviations, or confidence intervals are reported in the registry record.

The endpoint is described as a ratio, but the record does not clarify whether the named mean and standard deviation apply to Ct values, expression values, or a calculated ratio. It does not report transformations, handling of values outside assay limits, or how observations across laboratory conditions were combined. These details should not be inferred.

What is multiplicity, and why can it matter?

In general, multiplicity arises when several outcomes, genes, stimuli, time points, or group contrasts are tested. The more comparisons made, the greater the chance that at least one result appears unusual by chance if each is interpreted separately. Statistical plans may address this by specifying a primary contrast or adjusting inference across tests. The registry describes multiple genes, stimuli, and time points, but does not report a multiplicity strategy or a detailed analysis plan. It is therefore not possible to determine how any collection of comparisons was intended to be interpreted.

What can a planning calculation say?

In general, a sample-size calculation is a planning tool that describes the size of a future study under chosen assumptions. It is not a measure of the probability that an observed result is true, and it is not observed power. The registry's planning illustration is described as using a two-sided significance level of 0.05, equal allocation, a normal approximation, and a fraction of the observed effect; it distinguishes participants per arm for ordinary outcomes from total events for time-to-event outcomes. Such an illustration is not the trial's own design justification. A sample-size justification and the statistical plan for this study are not reported in the registry record.

The record's status as completed does not substitute for estimates or an analysis. Without reported outcome summaries, the stated endpoint, group structure, and measurement method can be described, but treatment effects, precision, and statistical evidence cannot be evaluated.

10. Multiplicity, Missing Data and Analysis Populations

The registry record lists three outcome measures and zero analyses. It does not report primary outcome estimates or describe the statistical analysis plan. The endpoint involves two genes, several in-vitro stimuli, and measurements at different time points. In general, examining many outcomes or contrasts raises a multiplicity concern because isolated findings may occur by chance. Whether the study specified a primary contrast, adjusted for multiple comparisons, or treated other outcomes as exploratory is not reported in the registry record.

Missing-data handling is also not reported in the registry record. The summary describes planned blood collections at baseline and follow-up for the frequent-outbreak group, but does not state whether all scheduled samples were obtained, how incomplete measurements were handled, or whether any participants were excluded from analyses. The enrollment figure and group recruitment description do not establish the number of usable observations for each endpoint, stimulus, or time point.

The analysis populations are not reported in the registry record. It is not stated whether analyses included everyone enrolled, only those with available laboratory measurements, or another defined set. No approach to assay failures, protocol deviations, or missing follow-up measurements is described.

These omissions limit interpretation independently of the study's non-randomized design. Without a stated analysis population, missing-data approach, and multiplicity plan, readers cannot determine which observations contributed to an estimate or how a set of comparisons was interpreted. Since the record also contains no reported analyses, these issues cannot be evaluated from outcome results.

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's adverse-event figures list the group titled “6 or More Herpes Labialis Outbreaks.” For that group, 12 were at risk for serious adverse events, zero were reported as affected by serious adverse events, and zero deaths were reported. The record also lists 12 at risk for other adverse events and three affected. These are the figures recorded for the listed group; the record does not provide serious-event terms.

In general, “serious” and “other” adverse events are separate reporting categories. A count in the other-events category should not be treated as a serious-event count. The listed figures do not establish the nature, timing, severity, or relatedness of the other events, and the record does not identify their terms. It also does not provide a comparative safety analysis against the other outbreak-history groups.

These figures should be read as descriptive registry reporting, not as proof that no serious events could occur. The information applies to the group and reporting recorded in the registry; it does not establish the completeness of event ascertainment or support a broader safety conclusion. The record does not report additional safety analyses.

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

The registry record describes an exploratory phase I investigation of immune measures in people with HSV-1 infection and different histories of herpes labialis outbreaks. It identifies laboratory assays for PBMC proliferation, immune-related gene expression, and serum anti-HSV-1 IgG. It also describes a single topical SADBE application for the frequent-outbreak group and follow-up blood collection for that group.

What the record does not establish is the direction or size of a change in IFNG or IL5 expression, whether any group differences were statistically distinguishable, or how precisely an effect was estimated. No primary outcome estimates, confidence intervals, p-values, or analysis details are reported in the registry record. Thus, the posted information does not support a conclusion about the measured immune response after SADBE.

Even if outcome estimates were available, the study structure would require careful interpretation. The groups were formed according to outbreak history, allocation was non-randomized, and the intervention was given to the frequent-outbreak group. Differences between that group and the others could reflect baseline group characteristics as well as treatment exposure. A before-and-after comparison in the treated group would describe change over time, but would not on its own identify the cause of that change.

These laboratory endpoints also should not be equated with clinical outcomes. The registry describes measurements of gene expression, cell proliferation, and antibody levels; it does not report results showing changes in outbreak frequency or other clinical outcomes. The study description can clarify what was measured and when, but the absence of reported analyses means the record cannot answer whether the measurements changed or what their clinical relevance might be.

Overall, the study record is useful for understanding the planned immune assessments and the non-randomized design. It is not a basis for claiming that SADBE does or does not alter immune responses, or for drawing medical conclusions.

13. Why This Trial Matters Statistically

This study illustrates how a trial can combine a treatment exposure with observational comparisons. The frequent-outbreak group received SADBE, while the other groups were defined by their outbreak histories. Such a design makes it important to distinguish a description of group differences from an estimate of an intervention effect. Non-randomized allocation does not ensure that groups differ only in treatment exposure.

It also highlights the importance of aligning the endpoint, time points, and comparison being analyzed. The primary endpoint title refers to an expression ratio between eight weeks and baseline, while the population and timeframe descriptions refer to day 57 for the frequent-outbreak group and day 1 measurements for the other groups. The registry record does not resolve these differences in wording. Clear definitions are needed to know which observations form the intended contrast.

The laboratory plan includes two genes, multiple stimuli, and repeated measurements. In general, this breadth can be useful for characterizing immune responses, but it also makes a prespecified analysis strategy important. Without a reported multiplicity plan and outcome estimates, readers cannot distinguish a primary finding from exploratory patterns or assess uncertainty.

Finally, the record shows why completion status and design details are not substitutes for results reporting. The study is listed as completed, but no analyses are listed. Without analyzed outcomes, the intended immune questions cannot be evaluated statistically from the registry record. This distinction matters when interpreting early-phase research: a detailed measurement plan can describe what investigators set out to assess, while conclusions require reported data and transparent analyses.

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.

The principal limitation for interpreting outcomes is that the registry record lists zero analyses and reports no estimates for the primary endpoint. The endpoint title, timeframe, and population description do not align in a fully clear way: they refer to an eight-week comparison, day 57 for the frequent-outbreak group, and day 1 for the other groups relative to the frequent-outbreak group's baseline. The exact intended contrast is not clarified.

Group membership was based on self-reported outbreak history, and allocation was non-randomized. SADBE was applied only to the frequent-outbreak group in the study description. Consequently, treatment exposure is entangled with group definition, and comparisons cannot be interpreted as randomized treatment contrasts. Although the registry says the other groups were matched to the frequent-outbreak group on demographic characteristics, matching details are not reported in the registry record.

The record does not describe the statistical analysis plan, analysis populations, handling of missing data, or multiplicity strategy. It does not state how laboratory measurements from different stimuli were combined or how the stated ratio was derived from Ct measurements. No outcome-level sample counts or variability estimates are reported.

Safety information is limited to the listed adverse-event figures for the frequent-outbreak group, with no serious-event terms and no comparative safety analysis reported. The registry describes laboratory and antibody measures but does not provide outcome results or clinical outcome analyses. These limitations mean the record supports a description of study structure and measurement intent, not an assessment of efficacy, immune effects, or comparative safety.

15. Related Tutorials

16. Related Statistical Calculators

17. Sources

• ClinicalTrials.gov: Immune Response and General Immune Health in Subjects Infected With Herpes Simplex Virus Type 1 (HSV-1) — NCT03661541.
• Registry record last updated 2021-11-03; status last verified 2021-10.
• Results first posted 2021-11-03.
• 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 lists NCT03661541 as a completed phase I, parallel, non-randomized study without masking. Its enrollment is listed as 36. Participants were grouped by self-reported herpes labialis outbreak history, and the frequent-outbreak group received a single topical application of 2% SADBE in DMSO after baseline blood collection.

The study description covers PBMC proliferation and gene expression after in-vitro stimulation, plus serum anti-HSV-1 IgG. The primary endpoint concerns IFNG and IL5 expression measured by qRT-PCR Ct. The registry lists three outcome measures but zero analyses, and does not report primary outcome estimates, uncertainty, or a statistical analysis plan. The listed safety figures for the frequent-outbreak group include zero affected by serious adverse events and zero deaths, with three affected by other adverse events.