AeroVax: study protocol for a randomised, double-blind, placebo-controlled, phase 2 trial to evaluate safety and immunogenicity of a next-generation COVID-19 vaccine delivered by inhaled aerosol to humans

Abstract

Background

Coronavirus disease 2019 caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) continues to have a significant impact worldwide, in part due to a reduction in neutralising antibody protection provided by vaccines targeting monovalent spike antigens related to immune escape. Development of vaccines amenable for respiratory mucosal delivery that provide broad and durable immunity are needed. This study aims to determine the safety and immunogenicity of a single inhaled dose of a replication-deficient chimpanzee adenovirus type 68 vector expressing a trivalent transgene cassette of SARS-CoV-2 S1 domain of spike, nucleocapsid and RNA polymerase genes (ChAd-triCoV/Mac).

Methods

We plan to recruit 350 nonpregnant adults aged 18–65 who have previously received three intramuscular SARS-CoV-2 mRNA vaccines in this 24-week, multicentre, double-blind, parallel-group, phase II, two-sided superiority randomised controlled trial. Participants will be randomised 2:1 to receive either a single inhaled dose of ChAd-triCoV/Mac or placebo, both aerosolised via the AeroNeb® Solo vibrating mesh nebuliser. A subset of separately randomised participants will undergo bronchoalveolar lavage (BAL). The co-primary outcome to be analysed in the per-protocol population is SARS-CoV-2 antigen-specific CD4⁺ and CD8⁺ T-cell responses measured at 2 weeks in the peripheral blood; solicited adverse event frequency to day 7 and unsolicited to day 28. In the BAL sub-study, the co-primary outcome will also include BAL fluid SARS-CoV-2 antigen-specific CD4⁺ and CD8⁺ T-cell responses measured at 4 weeks.

Conclusion

This placebo-controlled phase 2 randomised trial will test the safety and immunogenicity of an inhaled, nebulised ChAd-triCoV/Mac SARS-CoV-2 vaccine that is novel in its administration route and targeting of multiple viral epitopes. The results will provide further information regarding the mucosal T-cell response to immunisation.

Shareable abstract

We outline a planned randomised, double-blind, placebo-controlled phase 2 trial to evaluate safety and immunogenicity of a single inhaled dose of a replication-deficient chimpanzee adenovirus vector expressing multivalent SARS-CoV-2 antigens https://bit.ly/4jq9z7e

Introduction

Background and rationale (SPIRIT 2013 items 6a and 6b)

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to have a profound impact worldwide, with over 7 million deaths reported to the World Health Organization up to July 2024 [1]. COVID-19 continues to disproportionately affect under-represented and marginalised communities [2], posing an ongoing risk due to its ability to mutate, improve its transmissibility and evade immune responses [3]. While vaccination remains the most effective strategy to control COVID-19 by significantly decreasing hospitalisation and mortality rates [4], SARS-CoV-2 variants of concern (VOCs) that have evolved from ancestral SARS-CoV-2 have routed efforts to control breakthrough infections by eroding the protection afforded by their neutralising antibody-mediated immunity [5–8]. Current vaccine platforms targeting monovalent SARS-CoV-2 spike antigens must thus tailor the spike antigen expressed to the circulating strain, constraining the long-term feasibility of this approach [9–14]. Current first-generation vaccine strategies also pose a major immunological gap by their lack of induction of a protective early lung mucosal immune response, resulting in an ongoing risk of breakthrough infection in immunised persons over time and waning of vaccine-induced antibody responses from the lack of a sustained mucosal T-cell response [12, 15–20].

The development of alternative strategies for boost immunisation is one way to react to the dearth of vaccines providing more durable protective immunity against SARS-CoV-2 [9, 21].

The use of a recombinant viral-vectored multivalent vaccine administered via the respiratory route may offer one such solution [15, 16, 18, 19, 21] that is novel in its approach. By expressing highly conserved multivalent SARS-CoV-2 antigens, such aerosolised vaccines would induce potent T-cell responses in the lower respiratory mucosa absent from current intramuscular vaccination strategies, in a manner similar to SARS-CoV-2-infected patients with mild clinical disease [22, 23]. Strong T-cell-mediated responses have been demonstrated in individuals who are exposed to SARS-CoV-2 but test negative for infection, particularly against the β-coronavirus replication-transcription complex, suggesting cross-reactive T-cell immunity contributes to abortive infections [24].

While immune escape can occur with new VOCs due primarily to the antibody-mediated immunity from monovalent spike-directed vaccination, VOCs do not escape T-cell immunity due to the responses raised against largely conserved viral epitopes [25, 26]. Booster immunisation with a multivalent respiratory mucosal vaccine is therefore posited to induce a potent mixed immune response consisting of tissue-resident CD4/CD8⁺ T-cells, mucosal antibodies and trained innate immunity. As opposed to intranasal vaccination, which primarily targets the upper respiratory tract and results in limited mucosal immunity, inhaled aerosol vaccines primarily target the lower respiratory tract [27, 28] and have been shown in viral-vectored tuberculosis vaccine trials to induce tissue-resident T-cell and trained alveolar macrophages. Aerosol delivery through a mouthpiece has the added benefit of bypassing the nasal passage, decreasing the risk of retrograde infection to the olfactory bulb [29].

In a murine model, trivalent adenoviral-vectored SARS-CoV-2 vaccines expressing SARS-CoV-2 S1 domain of spike (S1), nucleocapsid (N) and truncated RNA polymerase (POL) genes provided the best protection against ancestral SARS-CoV-2 and VOCs when administered via the respiratory route compared with the intramuscular route [15]. In these preclinical studies, we found that trivalent vaccines performed better than bi- or monovalent counterparts, and that the use of a chimpanzee origin adenoviral backbone (ChAd-triCoV/Mac) provided superior protection to a human adenoviral-derived one (HuAd-triCoV/Mac) [15]. In a phase 1 clinical study, we tested single aerosolised escalating-dose delivery of both HuAd-triCoV/Mac and ChAd-triCoV/Mac, in both SARS-CoV-2-infected and -uninfected participants with previous SARS-CoV-2 messenger RNA (mRNA) vaccination history [30]. All vaccine dosage forms tested were safe and well tolerated; however, ChAd-triCoV/Mac achieved a more potent induction of dose-dependent respiratory mucosal T-cell response, with a smaller concomitant increase in airway antibody responses [30].

Based on these findings, we hypothesise that a single, optimal aerosolised dose of ChAd-triCoV/Mac will induce potent increases in SARS-CoV-2-specific T-cell responses in the respiratory mucosa with a favourable safety profile when compared with placebo.

Objective (SPIRIT 2013 item 7)

The primary objective is to determine the safety and immunogenicity of a single-dose aerosol delivery of the trivalent ChAd-triCoV/Mac SARS-CoV-2 vaccine in participants previously vaccinated with at least three intramuscular doses of a Health Canada-approved mRNA-based SARS-CoV-2 vaccine, either with or without previous SARS-CoV-2 infection history.

Methods: participants, interventions and outcomes

Trial design (SPIRIT 2013 item 8)

The AeroVax study is an investigator-initiated, phase 2, multicentre, double-blind, parallel-group, superiority-powered randomised placebo-controlled trial in Canadian adults aged 18–65 years.

Study setting (SPIRIT 2013 item 9)

Eligible participants will be identified from Canadian academic hospital clinical trial sites in Hamilton, Halifax and Ottawa. A subset of participants eligible for and enrolled in the main trial at the McMaster University site will be consented for a sub-study requiring bronchoalveolar lavage (BAL) that involves additional eligibility criteria and investigations, and whose participants will be subject to additional outcome measures. Participants who decline BAL sub-study consent and those participants who fail screening criteria for the BAL sub-study will remain eligible to participate in the main study. Participants at study sites in Halifax and Ottawa will only be eligible to enrol in the main study.

The study was prospectively registered with the United States National Institutes of Health (NCT06381739). This trial protocol is reported according to the SPIRIT and SPIRIT-Outcomes 2022 guidelines (supplementary materials S1) [31, 32]. Regulatory approval for the use of ChAd-triCoV/Mac SARS-CoV-2 vaccine in this clinical trial was granted by Health Canada (control no. 284864) and the Clinical Trials Ontario research ethics board (project ID 4846). Patient enrolment is planned to begin in January 2025.

Eligibility criteria (SPIRIT 2013 item 10)

Eligible participants agree not to enrol in any other intervention studies for the duration of the trial, where the other interventions could be reasonably expected to be associated with adverse events (AEs) overlapping with those expected of ChAd-triCoV/Mac or the immune responses being measured. A summary of the inclusion and exclusion criteria is found in table 2.

TABLE 2.

Summary of inclusion, exclusion, stopping and termination criteria for the phase 2 AeroVax trial

Inclusion criteria
All study participants	Additional criteria for bronchoalveolar lavage (BAL) sub-study participants only
18–65 years old on the randomisation date.	Normal complete blood count and creatinine at randomisation.
Received at least three doses of an mRNA SARS-CoV-2 vaccine.	Forced expiratory volume in 1 s (FEV1) and an FEV1/forced vital capacity (FVC) ratio greater than the lower limit of normal at randomisation.
Negative pregnancy test prior to intervention and be willing to use an effective contraceptive method for 8 weeks post-intervention, if of childbearing potential.
Read, write and communicate in the English or French languages.
Understand and comply with protocol requirements and instructions.
Exclusion criteria
All study participants	Additional criteria for BAL sub-study participants only
Receipt of any recombinant adenoviral-vectored SARS-CoV-2 vaccine (e.g. Vaxzeria® or Jcovden®).	Medical contraindication to bronchoscopy as determined at randomisation.
SARS-CoV-2 infection in the past 90 days confirmed by lab-documented or self-reported PCR or antigen test.	Taking anticoagulant medications.
Receipt of any SARS-CoV-2 vaccine within 90 days, or any other vaccine within 2 weeks, prior to study entry.
Receipt of any monoclonal antibodies for coronavirus disease 2019 (COVID-19) treatment within the preceding 3 months.
History of severe adverse reaction to previous COVID vaccination.
Known allergy to vaccine components.
Potential contraindication to SARS-CoV-2 vaccination (e.g. venous or arterial thrombosis with thrombocytopenia after vaccination, cerebral venous thrombosis with thrombocytopenia, heparin-induced thrombocytopenia, myocarditis or pericarditis).
Enrolment in any other COVID-19 experimental trial within the previous 90 days.
Pregnant or breastfeeding at randomisation.
Living with HIV infection and have a self-reported or laboratory-confirmed viral load >20 copies·mL−1. There is no concomitant per cent CD4+ T-cell count exclusion criterion for persons with HIV.
Active pulmonary disease at randomisation (e.g. asthma, chronic bronchitis, interstitial lung disease, pulmonary hypertension, lung cancer, cystic fibrosis, bronchiectasis).
Current use of daily inhaled corticosteroids for any medical condition.
Moderate or severe immunocompromise (e.g. transplant recipients, receipt of CAR T-cell therapy, current receipt of chemotherapy for underlying malignancy, receipt of rituximab, receipt of >30 mg prednisone equivalent daily of corticosteroids; or have a moderate or severe primary immunodeficiency syndrome).
Trial stopping (pausing) rules
Adverse events (AEs)	>25% of participants within first 28 days following vaccination report ≥Grade 3 AE.
Laboratory AE	Laboratory abnormality ≥Grade 3 persisting for >72 h, in >25% of participants following vaccination.
Spirometry	FEV1 or FVC decreases to ≤60% of predicted, in >25% of participants following vaccination.
Serious adverse event (SAE)	SAE is possibly, probably or definitely related to vaccination.
Trial termination rules
Incidence of AEs in this, or any other, trial using a similar vaccine vector or administration route indicates a possible health risk to participants.
New scientific knowledge becomes available that obviates clinical equipoise.

Trial stopping rules may be triggered by the Principal Investigator or Data Safety Monitoring Board (DSMB), whereas trial termination rules may only be triggered by the DSMB or Health Canada regulatory authority.

Intervention(s) (SPIRIT 2013 item 11)

Eligible participants will be randomised 2:1 to receive either a single 6×10⁷ tissue culture infectious dose 50% (TCID⁵⁰) of replication-deficient chimpanzee adenovirus type 68 vector expressing a trivalent transgene cassette consisting of the S1 region of the SARS-CoV-2 spike, full-length N and a highly conserved truncated region of the RNA POL genes (ChAd-triCoV/Mac) diluted in 0.5 mL formulated buffer; or a single 0.5 mL formulated buffer placebo dose. Both intervention and control will be administered using the well-characterised AeroNeb® Solo vibrating mesh nebuliser over two minutes of tidal respiration. No intervention dose modifications will be permitted, and intervention administration will be directly observed by blinded study personnel to ensure adherence. Following the intervention, participants will be advised to defer any routine vaccinations for 2 weeks and any SARS-CoV-2 booster vaccinations for 8 weeks. Any other medications taken by participants for 28 days following vaccination will be documented.

Outcomes (SPIRIT 2013 item 12, SPIRIT-outcomes 2022 items 12.1–12.5)

The co-primary outcome includes a primary immunogenicity outcome and a primary safety outcome; we chose this approach to demonstrate the safety and immunogenicity of the vaccine in the population of interest and inform correlates of protection in the lung.

The primary immunogenicity outcome is the change from baseline in the frequency of S1, N and POL SARS-CoV-2 antigen-specific CD4⁺ and CD8⁺ T-cell responses measured at 2 weeks in the peripheral blood. In the main study, the vaccine will be considered superior to placebo if it increases the number of circulating antigen-specific CD4⁺ and CD8⁺ T-cell responses in blood exceeding the baseline values by at least 2 sd. The primary safety outcome is a composite of the frequency, incidence and nature of grade 3, 4 or 5 AEs that are possibly or probably related to intervention up to week 24; the frequency of solicited AEs to day 7; and the frequency of unsolicited AEs to day 28.

In the study subset who undergo BAL, the co-primary outcome's immunogenicity outcome will also include S1, N and POL SARS-CoV-2 antigen-specific CD4⁺ and CD8⁺ T-cell responses measured at 4 weeks in the BAL fluid. In the BAL sub-study, the vaccine will be considered superior to placebo if it produces a minimum 10-fold (1 log₁₀) increase in antigen-specific CD8⁺ T-cell responses in the BAL fluid. The timing of the additional BAL outcome measures was chosen to reflect the optimal mucosal immune response seen in our phase 1 study [30]. The BAL sub-study's primary safety outcome will be assessed as in the main study participants.

Secondary outcome measures include: any reverse transcriptase PCR (RT–PCR)-confirmed SARS-CoV-2 infection to week 24; neutralising and total antibody levels in the blood at 2, 4 and 8 weeks; tissue-resident memory surface marker expression of CD103 and CD69 by airway T-cells at 4 weeks post-intervention; CD4 and CD8 cell responses specific for S1, N and POL SARS-CoV-2 antigens in the peripheral blood, including those expressing memory T-cell markers, at 4 and 8 weeks post-intervention; and the frequency of any AEs to week 24. These secondary outcomes were chosen to assess antigen-specific memory T-cells and their longevity in the mucosa and peripheral blood. SARS-CoV-2 infection testing will occur if participants are symptomatic, using a participant self-collected oral-nasal swab for SARS-CoV-2 RT–PCR testing.

In the BAL sub-study, additional secondary outcome measures include neutralising and total antibody levels in the BAL fluid at 4 weeks, and antibody responses against SARS-CoV-2 spike and receptor-binding domain (RBD) proteins in saliva at 2, 4 and 8 weeks. Antibody measurements in the sub-study participant BAL fluid and saliva will include measurements of IgA responses. These outcomes were chosen to demonstrate site-specific immunogenicity of the intervention.

Participant timeline (SPIRIT 2013 item 13)

The participant outline is found in figure 1, while a summary figure of major investigations and procedures is found in figure 2.

FIGURE 1.

Schedule of enrolment, interventions and assessments for the phase 2 AeroVax trial. BAL: bronchoalveolar lavage; β-hCG: β-human chorionic gonadotropin; CBC: complete blood count; ALT: alanine transaminase; ALP: alkaline phosphatase; POL: polymerase; RBD: receptor-binding domain; S: spike. Investigators may deviate ±3 days from scheduled dates for 2- and 4-week visits and ±7 days for 8-, 12- and 24-week visits. ^#: Screening should be performed within 28 days of the baseline visit. ^¶: The allocation visit can occur on the same day as the screening visit. ⁺: Virtual visits by phone, video or e-mail. ^§: Physical examination may occur at either the enrolment or allocation visit. ^ƒ: Adverse events will be solicited to day 7, unsolicited to day 28. Serious adverse events and medically attended adverse events will be documented for the duration of the study. ^##: For study participants of childbearing potential only. May be repeated at investigator discretion if suspicion of pregnancy. ^¶¶: Testing listed may be done at either the enrolment or allocation visit, unless a participant is enrolled in the BAL sub-study in which case such investigations will be carried out only at the enrolment visit. ⁺⁺: Blood sample for the allocation visit only may be drawn between −7 and 0 days.

FIGURE 2.

Graphical summary of major investigations and procedures for the phase 2 AeroVax trial. BAL: bronchoalveolar lavage.

Sample size (SPIRIT 2013 item 14, SPIRIT-outcomes 2022 item 14.1)

Given the novelty of the vaccine trial's co-primary outcome, no data exist to inform the minimal important difference in T-cell responses for both BAL fluid and blood. Based on the T-cell responses observed in our phase 1 trial [31, 32], we estimate a sample size of 350 participants will enable the detection of a small-to-medium treatment effect size (Cohen's d=0.4) with estimation of AE rates with a margin of error of 0.01 using a 95% confidence interval. In the event of slow participant recruitment, using the aforementioned assumptions, a sample size of 205 participants would enable the detection of a medium treatment effect size (Cohen's d=0.5). To detect a large effect size (Cohen's d=0.719) with 95% power, 68 participants in the vaccine group and 34 in the placebo group would be required, corresponding to detection of a mean per cent difference in antigen-specific T-cells of 0.739%. This calculation presumes a mean percentage of antigen-specific T-cells in peripheral blood of 1.051% (σ=1.441) and 0.312% (σ=0.188) in the intervention and control groups, respectively. We calculated the sample size using a one-sided Mann–Whitney U-test of the null hypothesis that there is no difference between intervention and control groups. An illustration of the projected sample sizes based on Cohen's d effect size detected is shown in figure 3.

FIGURE 3.

Range of sample size estimates by immune response in the vaccination group for the phase 2 AeroVax trial. Sample size calculations performed based on a one-sided Mann–Whitney U-test of the null hypothesis, with p<0.05. Vaccine effect size refers to the Cohen's d.

A sample size of 60 (allowing for 15% dropout) within the BAL sub-study will be adequate to report a range of significant correlation coefficients as described above for the main study.

Recruitment (SPIRIT 2013 item 15)

Each study site will screen and enrol participants until the total sample size is achieved. A combination of research databases, advertising in print and social media approved by local research ethics boards (REBs), and community outreach to populations typically under-represented in clinical trials, including Black and Indigenous persons, will be used to recruit participants.

Methods: assignment of interventions

Allocation (SPIRIT 2013 items 16–17)

Participants will be allocated using a computer-generated sequence in permuted blocks of random sizes, stratified and blocked by study site. Details of planned blocking are provided in a separate document unavailable to those who enrol participants or assign interventions. Once enrolled in the BAL sub-study, eligible participants in the BAL sub-study will be randomised separately from participants in the main study. Randomisation lists were generated by the trial statistician independently of the investigators with the ralloc command in Stata v.18.0 (StataCorp, College Station, TX, USA), using Research Electronic Data Capture (REDCap) tools to maintain central allocation concealment.

Participants and investigators (including clinical trial staff, outcome assessors and staff performing laboratory assays) will be blinded to treatment assignment. Only the staff at an external logistics facility labelling the vaccine, the statistician preparing the randomisation lists and the Data Safety and Monitoring Board (DSMB) will not be blinded to treatment assignment.

Unblinding will be permissible only for emergent medical management of study participants. Emergency unblinding requests will be reviewed by the blinded principal investigator (PI) or designate, after being submitted to the Coordinating Centre Trial Research Coordinator (CCRTC) or designate. In the event that the unblinding request is approved, the CCRTC will contact the unblinded study statistician and provide the information requested to the site investigator. Site investigators will be instructed to only share the treatment allocation with members of the participant's medical team if it is deemed medically necessary.

Methods: data collection, management and analysis

Data collection methods (SPIRIT 2013 items 18–19, SPIRIT-outcomes 2022 items 18a.1, 18a.2)

Participants will report relevant data to study investigators, which will be collated into a centralised REDCap trial database using case report forms and screening logs. The data from an online or paper-based diary kept by participants to record their oral temperature and any symptoms experienced during 7 days post-vaccination (figure 1) will also be entered into the relevant REDCap forms. All participant data entered into REDCap will be coded using a unique participant number. It will be validated for completeness by a central data manager, with any queries resolved by discussion. Trial data will be retained in a manner concordant with Canadian regulatory guidelines.

Blinded study staff will assess the primary and secondary outcomes. Study participants will receive CAD 150 in compensation for attending vaccination and week 8 visits to maximise retention and follow-up completion. Bronchoscopy sub-study participants will receive an additional CAD 195 in compensation at completion of bronchoscopy, in line with local research policies.

Statistical methods (SPIRIT 2013 item 20a-c, SPIRIT-outcomes 2022 item 20a.1)

Descriptive statistics will be used to summarise demographic data as well as AE data by severity and relationship to vaccination using counts (percentage), mean (sd) or median (quartiles). The primary immunogenicity outcome will be analysed in the per-protocol population, defined as participants who received the intervention and have bloodwork drawn at the week 2 visit. Mean antigen-specific T-cell responses between study group primary outcomes will be analysed using the Mann–Whitney U-test, and antibody responses will be compared using geometric mean titres with either t-tests or Wilcoxon rank-sum squares, depending on the normality of the data. To mitigate any risks to participants, we will perform an interim safety analysis after the first 100 participants have been enrolled given the novelty of the vaccine. The primary safety outcome will be analysed in the per-protocol population, defined as participants who received the intervention. For the secondary immunological outcomes, the population is defined as any participant who received the vaccine and has immunogenicity results available. The secondary safety outcome will be analysed in the intention-to-treat population, defined as all enrolled participants.

In the BAL sub-study, a range of significant correlation coefficients ≥0.4 with 90% power and α=0.05 between lung and blood immune responses will be reported using the G power calculator for Pearson's correlation coefficients.

Adjusted analyses will be conducted for which a baseline antibody covariate will be included, using linear regression models, with correlations analysed using Pearson's rank coefficient test. T-cell and antibody responses in participants with baseline detectable anti-N antibody titres will be analysed separately, as such participants have previously had COVID infection. No imputation will be performed for missing data. If protocol nonadherence occurs, participants will be analysed as randomised. We will use two-sided statistical testing, with ɑ=0.05 level of significance. All analyses will be performed using Stata v.18.0 for Windows (StataCorp, College Station, TX, USA).

Methods: monitoring

Data monitoring (SPIRIT 2013 item 21)

We have convened a steering committee consisting of infectious diseases physicians, medical microbiologists, a respirologist, a clinical trials methodologist and research scientists. The steering committee will oversee critical logistics and trial progress, can communicate with the DSMB or participating sites as needed; and will be responsible for final manuscript preparation.

Stopping and termination rules for the trial are summarised in table 1. The DSMB and REBs will be notified in the event of stopping rule activation, in which case the DSMB, study sponsor and PI will conduct a safety review to determine whether trial enrolment may restart. The decision to terminate the trial may be made at any time and may be triggered only by the DSMB or on the advice of Health Canada.

TABLE 1.

Administrative information (SPIRIT 2013 items 1–5) for the phase 2 AeroVax trial

Title	AeroVax: study protocol for a randomised, double-blind, placebo-controlled phase 2 trial to evaluate safety and immunogenicity of a next-generation coronavirus disease-2019 (COVID-19) vaccine delivered by inhaled aerosol to humans
Brief title	AeroVax 1.0
Trial registration	NCT06381739, ClinicalTrials.gov
Protocol version	1.1
Protocol version date (YYYY-MM-DD)	2024–04-11
Protocol contributor and study principal investigator	Prof. Fiona Smaill, MB BCh MSc FRCPC Professor Emerita, Dept. of Pathology and Molecular Medicine, McMaster University smaill@mcmaster.ca
Central contact	Marilyn Swinton McMaster University 1280 Main Street West, Hamilton, Ontario, L8S 4L8, Canada swinton@mcmaster.ca
Trial sponsor name and contact information	McMaster University 1280 Main Street West Hamilton, Ontario, L8S 4L8 Canada
Role of study sponsor in design, collection, management, analysis, interpretation, report writing and publication submission	None
Funding	Canadian Institutes of Health Research Award no. 484280
Role of study funder in design, collection, management, analysis, interpretation, report writing and publication submission	None
Steering committee composition	Infectious diseases physicians, medical microbiologists, a respirologist, a clinical trials methodologist and research scientists.
Steering committee responsibilities	Overseeing critical logistics and trial progress, communicating with the data safety monitoring board or participating sites as needed; and responsibility for final manuscript preparation.
Data safety monitoring board composition	Three members, consisting of infectious diseases experts and clinical trial methodologists that are independent from the study sponsor or any competing interests.
Data safety monitoring board responsibilities	Meeting at regular intervals to review individual and cumulative data for adverse events and serious adverse events, providing advice to ensure the overall safety of participants and that adverse events documentation is in accordance with regulatory guidance, and performing two interim analyses during the trial for safety and immunogenicity.
Countries of recruitment	Canada
Condition(s) or focus of study	COVID-19 inhaled vaccine
Intervention: ChAd-triCoV/Mac	Intervention type: biological/vaccine Intervention name: ChAd-triCoV/Mac Intervention description: clinical-grade, fully certified ChAd-triCoV/Mac produced according to current Good Manufacturing Principles (cGMP) will be provided. A single dose of ChAd-triCoV/Mac diluted in 0.5 mL formulated buffer will be aerosolised and inhaled via a mouthpiece and tidal breathing over approximately 2 min using the AeroNeb® Solo Mesh Nebuliser.
Control	Intervention type: other Intervention name: control Intervention description: a single dose of placebo (0.5 mL formulated buffer) will be aerosolised and inhaled as the intervention vaccine.
Key inclusion criteria	1. Adults who are 18–65 years old on the day of randomisation. 2. Able to read, write and communicate using the English or French language. 3. Received at least three doses of an mRNA COVID vaccine. 4. Individuals of childbearing potential must have a negative pregnancy test prior to vaccination and be willing to practice an effective form of contraception for 8 weeks post-vaccination. 5. Agree not to enrol in any other intervention studies for the duration of the study, where the intervention could be reasonably expected to be associate with adverse events overlapping with the inhaled vaccine or the immune responses being measured. 6. Able to understand and comply with protocol requirements and instructions; able to report adverse events; able to attend scheduled study visits and complete required investigations. 7. For participants in the bronchoalveolar lavage (BAL) sub-study, haematology (complete blood count (CBC) and chemistry (creatinine)) within normal limits. 8. For participants in the BAL sub-study, forced expiratory volume in 1 s (FEV1)>the lower limit of normal (LLN) and FEV1/forced vital capacity ratio above the LLN.
Key exclusion criteria	1. Failure to provide informed consent. 2. Individuals who are pregnant or breastfeeding. 3. Have received any recombinant adenoviral-vectored COVID-19 vaccine (AstraZeneca (Vaxzeria®) or Johnson & Johnson (Janssen Jcovden®). 4. COVID infection (positive PCR or antigen test, self-reported or lab-documented) within the last 90 days. 5. Last dose of a COVID vaccine administered less than 90 days prior to study entry. 6. Administration of any vaccine within 2 weeks of study entry. 7. Active pulmonary disease diagnosed by a physician including asthma, chronic bronchitis, interstitial lung disease, pulmonary hypertension, lung cancer, cystic fibrosis or bronchiectasis. Current use of daily inhaled steroids for any condition. 8. Persons with HIV and a detectable HIV viral load (>20 copies·mL−1), self-reported or confirmed. There is no concomitant per cent CD4+ T-cell count exclusion criterion for persons with HIV. 9. Administration of monoclonal antibodies for treatment of COVID-19 infection within 3 months. 10. Moderately or severely immunocompromised (e.g. transplant recipients/CAR T-cell therapy, currently on chemotherapy for cancer or on potent immunosuppressant therapies such as rituximab or high dose steroids (>30 mg of prednisone equivalent daily) or moderate or severe primary immunodeficiency syndrome). 11. History of severe reaction to previous COVID vaccination (e.g. hives, difficulty breathing, high fever, seizures, myocarditis, pericarditis). 12. Potential contraindication to COVID vaccination (e.g. venous or arterial thrombosis with thrombocytopenia after vaccination, history of cerebral venous thrombosis with thrombocytopenia, history of heparin-induced thrombocytopenia, history of myocarditis or pericarditis). 13. Known allergy to vaccine components or previous receipt of any experimental adenovirus-vector vaccine by the aerosol route. 14. Enrolment in any clinical trial of experimental treatment for COVID infection within 90 days. 15. For participants in the BAL sub-study, any health-related condition for which study bronchoscopy is contraindicated. 16. For participants in the BAL sub-study, current use of anticoagulants.
Study design	Allocation: randomised Intervention model: parallel-group Primary purpose: prevention Phase: 2
Masking	Trial participants, investigators (including clinical trial staff, outcome assessors and staff performing laboratory assays).
Target sample size	350
Recruitment status	Not yet recruiting
Primary outcomes	Outcome: antigen-specific T-cell responses in blood. Timeframe: 2 weeks Outcome: antigen-specific T-cell responses in BAL sub-study. Timeframe: 4 weeks Outcome: any grade 3, 4 or 5 adverse events that are possibly or probably related to study vaccine. Timeframe: 24 weeks Outcome: frequency of solicited adverse events (AEs). Timeframe: 7 days Outcome: frequency of unsolicited AEs. Timeframe: 28 days
Secondary outcomes	Outcome: confirmed COVID infection by RT–PCR. Timeframe: 24 weeks Outcome: additional functional activities of CD4+ and CD8+ T-cells in peripheral blood. Timeframe: 8 weeks Outcome: neutralising and total antibody levels in BAL sub-study. Timeframe: 4 weeks

Harms (SPIRIT 2013 item 22)

We have convened a three-member DSMB consisting of infectious diseases experts and clinical trial methodologists that are independent from the study sponsor or any competing interests. The DSMB will meet at regular intervals to review individual and cumulative data for AEs and serious AEs (SAEs), providing advice to ensure the overall safety of participants and that AE documentation is in accordance with regulatory guidance. All SAEs must be documented and reported within 24 h of occurrence to the CCRTC, who will inform the Health Canada, relevant REBs and DSMB. We will use modified Common Terminology Criteria for Adverse Events to assess severity and causality.

Based on our phase 1 study results [30] and the mucosal delivery of this investigational vaccine, we do not expect vaccine-induced thrombocytopenia; however, participants with a prior history of such complication after SARS-CoV-2 vaccination will be excluded from the trial (table 2), participants will be informed of this theoretical risk (supplementary text S2), and a complete blood count will be obtained at the enrolment/allocation, 2-, 4- and 8-week visits to mitigate potential harm to participants (figure 1).

The DSMB will perform two interim analyses during the trial: in the main trial, an interim analysis of safety outcomes will occur once 100 participants have been enrolled; in the BAL sub-study, unblinded data from the first 15 participants at the 4-week timepoint will be reviewed for immunogenicity outcomes. For the immunogenicity interim analysis, a mean increase of ≥20% in spike-elicited CD8⁺ T-cells in >40% of study participants is expected based on phase 1 study results [31, 32]. Any unexpected interim analysis results will trigger discussion with the PI and study sponsor regarding next steps.

Auditing (SPIRIT 2013 item 23)

A monitor independent from the investigators and sponsor will conduct an inspection of each trial site prior to the inclusion of the site's first participant. The monitor will conduct regular quality control inspections to ensure that the trial's eligibility criteria are being applied, standard operating procedures for informed consent and other trial processes are being adhered to, case report forms are being accurately completed, and there is compliance with SAE reporting and vaccine management principles. The monitor will also conduct a site visit at the end of the study to ensure accountability for all trial products and documentation.

Methods: ethics and dissemination

Research ethics approval (SPIRIT 2013 item 24)

Application was made to and approved by the Hamilton Integrated Research Ethics Board acting as the board of record for Clinical Trials Ontario (project no. 4846). Application will be made to the Research Ethics Board at Dalhousie University. The study will be conducted in accordance with applicable Canadian regulations, the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use guidelines on current Good Clinical Practice, the Tri-Council Policy Statement on Ethical Conduct for Research Involving Humans and the Declaration of Helsinki.

Protocol amendments (SPIRIT 2013 item 25)

Protocol amendments will be communicated to Health Canada and relevant REBs according to national reporting standards. Upon receipt of REB approval for modifications, the trial registration record will be amended on ClinicalTrials.gov. Protocol amendments relevant to participants, such as consent form modifications, will be communicated at the next study visit by telephone or e-mail.

Consent or assent, and confidentiality (SPIRIT 2013 items 26–27)

Signed informed consent will be obtained in person before the first study visit by study staff at the participating site. At the initial visit, participants who provide consent at the McMaster University site will be informed of the BAL sub-study and, if interested in participating, a second signed informed consent specific to the sub-study will be obtained. In accordance with Canadian federal regulations, participants’ personal information will be encrypted and de-identified through the use of a study key; identifiable data would only be shared with explicit consent of participants or as required by law.

Access to data (SPIRIT 2013 item 29)

The final trial dataset will be accessible to the PI, trial statistician and DSMB; no contractual agreements exist that limit such access. For the template consent form that will be used in the trial, see supplementary materials S2.

Ancillary and post-trial care (SPIRIT 2013 item 30)

Not applicable.

Dissemination policy (SPIRIT 2013 item 31)

Authors of the phase 2 clinical trial manuscript will be determined by the PI and must meet the International Committee of Medical Journal Editors criteria. We do not intend to use professional writers in the manuscript drafting process. Trial results will be presented at national and international conferences and published as an open-access publication in a peer-reviewed journal within 24 months of study completion.

Discussion

This phase 2 trial will be the first randomised placebo-controlled trial of the multivalent adenoviral-vectored ChAd-triCoV/Mac SARS-CoV-2 vaccine administered by inhaled aerosol in adults aged 18–65 years who have previously received three intramuscular doses of a SARS-CoV-2 mRNA vaccine. Demonstration of successful induction of SARS-CoV-2-specific CD4/CD8⁺ T-cell responses across multiple viral epitopes in the lungs of participants may provide an indication that a more durable immune response to VOCs may occur compared with first-generation monovalent SARS-CoV-2 vaccines. In this trial, a subset of participants will undergo BAL for further characterisation of site-specific immune responses.

Our novel co-primary outcome will assess the safety and immunogenicity of ChAd-triCoV/Mac by evaluating S1, N and POL SARS-CoV-2 antigen-specific CD4⁺ and CD8⁺ T-cell responses measured at 2 weeks in the peripheral blood, and at 4 weeks in the BAL fluid in those study participants who undergo the procedure; as well as the AEs and SAEs. In light of this novel approach, our planned study recruitment is larger than the minimum sample size required to show a small-to-medium-sized effect in the trial to account for the lack of an accepted minimal clinically important difference (MCID) in T-cell responses. As a result, this trial's co-primary outcome may also help shed light on what the antigen-specific T-cell MCID in the lungs is in response to vaccination. The use of a placebo control in this trial is reasonable because all participants have been previously immunised with monovalent SARS-CoV-2 mRNA vaccines that have established clinical efficacy, and thus are not at risk of undue harm if they are randomised to receive placebo.

This investigator-initiated, phase 2, multicentre, double-blind, parallel-group, superiority-powered randomised placebo-controlled trial will be the first to test a multivalent adenoviral-vectored SARS-CoV-2 vaccine administered by inhaled aerosol in previously vaccinated adults aged 18–65 years. This study will help establish the safety of ChAd-triCoV/Mac and determine whether T-cell-mediated immunity can be directed against multiple viral epitopes, providing a more broad and robust immune response against ever-evolving SARS-CoV-2 variants.

Supplementary material

Please note: supplementary material is not edited by the Editorial Office, and is uploaded as it has been supplied by the author.

Supplementary material

00758-2024.SUPPLEMENT

Data availability

The unabridged research protocol, statistical analysis plan and/or de-identified individual participant data will be made available to qualified researchers upon written request to the corresponding author.