Serotype 3 Experimental Human Pneumococcal Challenge (EHPC) study protocol: dose ranging and reproducibility in a healthy volunteer population (challenge 3)

Study overview

This protocol has been reported following the SPIRIT reporting guidelines.19

This is a human challenge study of healthy adult participants who will be nasally inoculated with well-characterised, fully sequenced-penicillin sensitive SPN3, for the assessment of acquisition of nasal pneumococcal colonisation and immune responses. We will conduct a dose-ranging study to determine the optimum SPN3 isolate and dose for safe colonisation acquisition and confirm the dose and safety in a subsequent larger cohort in a reproducibility study. This study will run from July 2022 to October 2023 at the Accelerator Research Building, Liverpool School of Tropical Medicine (LSTM), Liverpool, UK.

Figure 1 displays the study process. In the dose ranging study, sequential cohorts of 10 healthy participants will be challenged with escalating doses of SPN3. We will start at 10 000 colony-forming unit (CFU)/naris and after n=10, escalate to 20 000 CFU/naris (n=10) and then 80 000 CFU/naris (n=10) if safe to do so. The first challenge cohort at 10 000 CFU/naris will start slowly with a smaller group (n=1–6) inoculated per week, for safety, before completing the group (n=10). We may increase the dose further depending on colonisation rates after consultation with the Trial Monitoring Group (TMG). If optimum attack/colonisation acquisition rates are achieved (≥50%) in the lower doses, the higher dose escalation groups may not be completed—this will be discussed with the trial steering committee (TSC) before a decision to omit the higher doses is made. We may escalate the dose before a cohort of n=10 is inoculated, and subsequent escalated doses may be omitted if the colonisation rates are low. This is in the interest of not inoculating participants at a dose that could be futile. This will be at the discretion of the chief investigator (CI).

Figure 1

Trial flow chart. *Isolate 2 may not be tested at the discretion of the trial steering committee. CFU, colony-forming unit; CI, chief investigator.

Up to 2 isolates will be tested in this manner, serotype 3 10V CC700 and serotype 3 LIV014-S3 CC180. In case of not achieving the desired attack rate with any of the isolates, the data obtained will be reviewed to either test an additional isolate or complete the cohort at a lower attack rate.

Using the dose, isolate and inoculation regime that results in ≥50% colonisation acquisition/attack rate, with a high safety profile, we will complete the cohort with another 33 participants to check for reproducibility of attack rate. In previous research conducted with SPN6B, colonisation rates of 58.5% were seen with single inoculation and of those who were negative after the first inoculation, a further 41.7% then became positive after a second inoculation, resulting in a combined colonisation rate following two inoculations of 70.7%.

Study objectives and outcomes

Study objectives and outcomes measures are demonstrated in table 1.

Study participants

Participant timeline

For both parts of the study, participants will attend an identical visit schedule; however, the samples taken at these visits will differ (figure 2). Participants will attend a screening visit, inoculation visit (day 0) and then follow-up visits at days 2, 7, 13 and 28. The screening visit should occur 5 days prior to inoculation but a window of −7/+4 days will be used. Figure 2 outlines the samples that are required at each visit.

Figure 2

(A) Dose-ranging study sampling schedule. (B) Reproducibility study sampling schedule. CPS, capsular polysaccharide; IgG, immunoglobulin G; SPN3, Streptococcus pneumoniae serotype 3. Created with Biorender.com.

All will first attend a screening visit to confirm eligibility through medical history, clinical examination and acquisition of samples including a full blood count and nasal wash sampling. Baseline nasal washes will be evaluated for natural colonisation with SPN (and serotype if present) through classical microbiological culture and molecular methods.

All participants will then attend an inoculation visit (day 0), where fluid containing SPN3 will be instilled into their nose. At this visit, they will be given a safety pack containing a thermometer, safety information leaflet and a 5-day course of amoxicillin. They will be instructed to contact the research team daily for 3–5 days with their temperature recording and any symptoms. Participants who report symptoms consistent with pneumococcal disease will be reviewed in person by a clinician and may be instructed to take their antibiotic course. Participants have 24-hour access to research clinicians as well as access to hospital facilities and prompt treatment if required.

In the dose-ranging study, a second targeted booster inoculation will be given to participants on day 14 if they have tested negative for SPN3 on day 2 and 7 samples. In the reproducibility study, it will be at the discretion of the CI to decide if targeted booster inoculation should be applied, based on results of the dose-ranging study. If a second inoculation is included in the reproducibility study, it will be only given at day 14 to those participants who are negative for SPN3 at days 2 and 7 (these participants will be asked to attend additional follow-up visits at days 16 and 21 for further sampling).

During the dose-ranging phase, nasal wash samples preinoculation and post inoculation will be collected to assess colonisation acquisition and density, by classical microbiological culture and molecular methods. Additionally, serum samples will be taken at baseline, 13 and 28-days after inoculation, to measure levels of anticapsule polysaccharide immunoglobulin G (IgG). Nasal cell samples will be taken to characterise cellular populations and dynamics by flow cytometry.

During reproducibility phase, nasal wash and nasal cell samples will be collected as in the dose-ranging study. A viral swab will be collected preinoculation to test for viral coinfection. Nasal filters (nasosorption) will be collected preinoculation and post inoculation to assess mucosal inflammation using 30-plex Luminex. Saliva samples will be taken preinoculation and post inoculation to compare rates, density and duration of SPN3 colonisation in saliva and nasopharyngeal samples. Peripheral blood mononuclear cells (PBMCs) will be obtained at baseline, at 13-days and at 28-days post inoculation. PBMCs will be used to characterise memory B cell and other immune cell populations using flow-cytometry based methods. Serum samples will be taken at baseline, 13 and 28 days after inoculation to measure levels of anticapsule polysaccharide IgG. Exhaled detection facemasks (EDF) will be collected at day 2, to allow comparison against other shedding samples for detection of SPN3. A subgroup of participants who have demonstrated colonisation with SPN3 at day 2 will undergo EDF sampling at day 7.

At the end of the study, study participants who have been positive for SPN3 colonisation at any time point, and who have not subsequently had two consecutive negative nasal wash samples, will be asked to take oral amoxicillin 500 mg three times daily for 5 days with the aim to clear/assist with clearing of colonisation.

Data collection methods

Samples will only be collected by staff members who are trained and delegated to do so. Table 3 describes the data collection methods.

Sample size calculation

We have adopted a stepwise approach to escalating the inoculation dose. The protocol is designed to: (1) minimise the possibility that we try repeatedly to attain colonisation at a dose in which it is unlikely to happen; (2) maximise safety by inoculating small groups before continuing onto larger groups (in which we will have the statistical power to give reasonable precision of our estimate of colonisation rates).

Based on previous experience in studies of SPN6B and SPN3, we expect that with a colonisation rate of 45%, with 95% confidence level, and a margin of error of 15%, our study will be complete 43 participants with a single inoculation dose. Depending on colonisation rates at different inoculum doses of the SPN3 isolate, we need a minimum of 43 and a maximum of 93 participants to complete the study. This allows for up to two isolates to be taken through to the highest dose of 80 000 CFU/100 µL. For example, if two isolates were tested at each dose in 10 participants in the dose ranging study, this would equate to 60 participants. A further 33 participants would then take part in the reproducibility study, equating to a total of 93 participants. To ensure that we complete the correct number of participants, we will over recruit to allow for screen failures and exclusion from primary outcome analysis for natural colonisation and loss of participants due to drop out. Based on the assumption that two isolates will be tested and estimating a 20% rate of drop out/screening failure, we will recruit a maximum of 117 participants. Participants who are natural SPN carriers will be included in analysis of exploratory outcomes.

Statistical analysis plan

This is an open label, non-randomised, safety and dose escalation study in healthy participants. Analyses are descriptive.

Timing of analysis

Data will be reviewed at the middle and the end of follow-up for each cohort of 10 healthy participants from the dose ranging phase as soon as database completion and lock occurs for that cohort. Data reviewed after day 7 will be for targeted booster inoculations if participants have tested negative for SPN3 on both days 2 and 7. Safety and outcome results will be reported to the TSC and TMG. Interim analysis in the dose ranging phase is to consider addition of second inoculation dose. Analysis at the end of each cohort is to consider dose escalation (or cessation). Once the dose has been selected, we will complete then reproducibility study and final analysis will be done on completion of that cohort after database completion and lock.

Analysis populations

The intention-to-treat (ITT) population is all participants who have been enrolled and who have received at least one inoculation dose (ITT). The main analysis population for each cohort is the modified ITT population, consisting of all participants who have been enrolled and received at least one inoculation dose (D0) and have had at least one valid outcome assessment measure (nasal wash). The safety analysis population is the ITT population and will include all participants who have received at least one inoculation dose.

Analysis will use available data. No imputation for missing endpoints will be performed.

Covariates and subgroups

If targeted booster inoculations are given, the numbers and results of these will be described, including a summary of colonisation after the second dose. Depending on the number receiving second doses, and on the endpoint being analyses, the results after the second dose will either be included as a ‘new’ inoculation event, potentially requiring adjustment for repeated measures, or will be excluded. Immunological data will be described, using appropriate estimators for the characteristics of the variable (arithmetic or multiplicative means, for example) and appropriate transformations as required. Where appropriate, and in particular for the reproducibility cohort, regression models will be used to estimate adjusted associations between strain and number of doses with colonisation as an outcome, and with immunological response as an outcome.

Interim analysis and data monitoring

Data will be reviewed on an ongoing basis for any safety or adverse events (AEs) and will be reported per protocol to the TSC and data and safety monitoring committee (DSMC). Efficacy outcomes will be evaluated after day 7 to provide information on second dose and at the cohort completion to assist with decision-making for the next dose escalation in sequence. The reproducibility cohort will have a single interim look, to evaluate second inoculation dose and a final analysis at the end of cohort follow-up.

Efficacy analysis

Each dose cohort (dose selection and reproducibility) will be analysed separately. Binary endpoints will be summarised as frequency (%) with 95% confidence estimates at each time point and by participant level summary. Density will be summarised using number, mean, geometric mean, SD, geometric SD, minimum and maximum at each time point. 95% CIs will be estimated for geometric mean estimates. Duration of colonisation (days) will be analysed as a continuous variable and summarised using mean, SD, minimum and maximum in the event of no censored outcomes. In the event of censoring, product limit methods will be used to estimate endpoints. The duration will be defined as the time between inoculation and the time of the last NW sample that is positive for colonisation. Symptom and sore through grading scales will be summarised by frequency (%). Agreement between colonisation results from microbiological culture and qPCR will be estimated using Bland-Altman plots and estimates of agreement.

The reproducibility cohort alone will use generalised linear models for single measurement outcomes (colonisation at any time vs not) under a binomial model with logit link to estimate odds ratio (95% CI) for strain/procedure/one or two doses as independent variables. Censored endpoints (time to colonisation) will be analysed using product limit estimates of median (95% CI for the time). Exploratory immunological outcomes will be summarised as above.

Reporting conventions

P values>0.0001 will be reported to 4 decimal places; p values<0.0001 will be reported as‘< 0.0001’. Distribution estimates such as mean, geometric mean, SD, median and quartiles will be reported to 3 decimal places. Parameters estimates such as regression coefficients, CIs and HRs will be reported to three significant digits.

Safety reporting

AEs will be graded using the Division of AIDS Table for Grading the Severity of Adult and Paediatric Adverse Events.20 If the severity of an AE could fall in either one of two grades, the higher of the two grades should be selected.

Symptoms experienced and attendance to hospital/GP will be asked about at each visit. Serious adverse events (SAEs) will be reported from the time of consent until completion of day 28 visit, or until completion of antibiotic treatment if participants require this.

All AEs will be recorded in the electronic case report form (eCRF) and documented in a weekly safety report. Adverse events of special interest (AESI) such as (but not limited to) headache, cough, sore throat and earache will be specifically documented and reported to the TMG, TSC and DSMC in the safety report.

All SAEs will be recorded on an SAE form and reported to the DSMC, sponsor within 24 hours of discovery or notification of the event. All SAEs/AESIs will be followed until resolution/stabilisation or until the end of the participants last study visit. The DSMC will perform an independent review of SAEs.

Auditing

A Trial Monitoring Plan will be developed by the Sponsor and agreed by the TMG and CI based on the trial risk assessment. Following written standard operating procedures, the monitors will verify that the clinical trial is conducted, and data are generated, documented and reported in compliance with the protocol, good clinical practice and the applicable regulatory requirements.

Ethics and dissemination

Consent

Potential participants will be sent a copy of the PIL (online supplemental material 1) and invited to contact a member of the team if they would like to participate. They will then be invited to attend a presentation and to carry out a quiz to ensure they have understood the information given. If a participant has voluntarily agreed to take part in the research and the study team are satisfied that they meet the eligibility criteria, they will be invited to provide written informed consent with a delegated, trained member of staff (online supplemental material 2). In line with recommended practice (MRC tissue and biological samples for use in research), participants will be asked to consent to gift their anonymised samples for use in future studies and shared with research collaborators and stored for any future commercial respiratory partnerships. This is outlined in the PIL and consent form.

Data management and participant confidentiality

Study data will be recorded directly into REDcap, an Electronic Data Capture system.21 Any additional information that needs recording but is not relevant for the case report form (CRF) will be recorded on a separate paper source document. The eCRF must be completed by designated and trained study personnel. Quality control will be performed on each eCRF. The processing of eCRFs will include an audit trail, to include changes made, reason for change, date of change and person making change.

Each participant will be assigned a unique, non-identifiable study number at recruitment for anonymisation. Unlinked non-identifiable clinical data will be stored and analysed at the LSTM, MSD or collaborating laboratories. Only authorised members of the clinical research team will be able to access participant personal information which is directly relevant to the study. All electronic records containing personal information will be stored in a password-protected database on a password protected computer. Paper documentation containing personal information will be kept in a locked filing cabinet in a locked room. On completion of the study, the eCRF will be locked and source documents will be photocopied and archived on paper and electronically in a secure database. This data will be stored for a minimum of 25 years. See online supplemental material 2 for information on storage of biological specimens.

Dissemination policy

The findings from this study will be disseminated among the scientific community. We intend to publish our findings in peer-reviewed scientific journals and present data at appropriate local, national and international conferences. In addition, we will produce a report of our findings, which will be made available to all participants. Authorship of the final trial report and subsequent publications will include those who contribute to the design, delivery and analysis of the trial. Authorship will be defined on study completion in line with International Committee of Medical Journal Editors guidelines.22

Regarding data sharing, we will hold exclusivity of participant data for 2 years, until all analysis and publications are complete. Deidentified individual participant data that underlie the results reported at the end of the trial will then be made available in an open format with accompanying metadata to the LSTM archive team, who can make it available on request. Researchers who provide a methodologically sound proposal will be able to request access, to achieve aims in the agreed proposal. Proposals may be submitted up to 5 years following publication of the results of this protocol. After 5 years, the data will be available in LSTM archives but without investigator support.

Patient and public involvement and engagement

This study is run in conjunction with the EHPC studies, which have been studying pneumococcal colonisation over the last 11 years. There are numerous opportunities for public and patient involvement: newsletters are sent out to all participants to inform them of the study results and further work, previous participants assist with recruitment events and social media accounts update followers about current studies and our ongoing work. For this study, we have asked participants from previous studies to review the PIL and consent form, to ensure it is clear and easy to understand.