A single dose of ChAdOx1 Chik vaccine induces neutralizing antibodies against four chikungunya virus lineages in a phase 1 clinical trial

Chikungunya virus (CHIKV) is a reemerging mosquito-borne virus that causes swift outbreaks. Major concerns are the persistent and disabling polyarthralgia in infected individuals. Here we present the results from a first-in-human trial of the candidate simian adenovirus vectored vaccine ChAdOx1 Chik, expressing the CHIKV full-length structural polyprotein (Capsid, E3, E2, 6k and E1). 24 adult healthy volunteers aged 18–50 years, were recruited in a dose escalation, open-label, nonrandomized and uncontrolled phase 1 trial (registry NCT03590392). Participants received a single intramuscular injection of ChAdOx1 Chik at one of the three preestablished dosages and were followed-up for 6 months. The primary objective was to assess safety and tolerability of ChAdOx1 Chik. The secondary objective was to assess the humoral and cellular immunogenicity. ChAdOx1 Chik was safe at all doses tested with no serious adverse reactions reported. The vast majority of solicited adverse events were mild or moderate, and self-limiting in nature. A single dose induced IgG and T-cell responses against the CHIKV structural antigens. Broadly neutralizing antibodies against the four CHIKV lineages were found in all participants and as early as 2 weeks after vaccination. In summary, ChAdOx1 Chik showed excellent safety, tolerability and 100% PRNT50 seroconversion after a single dose.

* ELISpot assays-Assays were ran on fresh cells, therefore samples could not be repeated. Responses were average across 3 technical triplicates and the mean response of the unstimulated (negative control) wells were subtracted. QC criteria were applied to samples before inclusion and analysis. For a sample to be considered valid, at least two technical replicates had to pass the pre-established QC parameters. We did not have any data point failing QC. Plates were counted using an AID automated ELISpot counter (AID Diagnostika GmbH, algorithm C) using identical calibration settings for all plates. Spot counts were adjusted only to remove artifacts.
* PRNT assays-Were initially ran as a single replicate for each biological sample, but validated by re-running around 30-40% of the samples in a second experiment. Internal positive and negative controls were included. Counting of plaques was performed by a single blinded operator. * Intracellular cytokine staining for flow cytometry-Experiments were ran on fresh cells, as a single replicate for each biological sample. QC criteria were applied to samples before inclusion and analysis. To maintain reproducibility between sample batches, the same cytometer and the same lot of antibodies were used for every run. Gating and analysis was performed by a single operator. We did not have any data point failing QC.
* ELISA assays -Were ran as 3 technical replicates for each biological sample. Each of the 96-well plates contained: a standard curve in duplicate, blank wells in triplicate and an internal control in triplicates. The internal control was prepared as an independent dilution to correspond to the 4th serial dilution of the standard curve. The standard curve OD values at 450 nm were fitted to a four-parameter model using the BioTek Gen5 v3.09 software. QC criteria were applied to the curve to ensure that the test samples were interpolated similarly between plates and that the coefficient of variance was low (<20%). Failure to meet these parameters resulted in failure of the assay and the whole sample plate was repeated. We did not have any data point failing QC.
This study was not randomized because required a dose-escalation approach and the IMP was not compared against placebo. To assess immune responses, each participant was compared to its own baseline prior to vaccination.
There was not blinding between the participants and the clinical team due to the type of study design (one product at dose-escalation). Scientists performing PRNT assays were blinded to the volunteer vaccination dosage group and time point. It was not possible to blind the ELISpot and ICS sample processing due to the fact that the samples had to be processed in fresh. Independent scientists from those performing the assays performed QC of all immunogenicity assays. Anti-human IFN-y, capture IgG1 mouse monoclonal Ab, clone 1-D1K (MABTECH, cat# 3420-3) -dilution 1:100. Anti-human IFN-y biotinylated , detection mouse IgG1 monoclonal Ab, clone 7-B6-1 (MABTECH, cat# 3420-6) -dilution 1:1000.
• Anti-human IgG (y-chain specific)-Alkaline phosphatase polyclonal antibody produced in goat (Sigma-Aldrich, cat# A3187). The manufacturer confirms on its website that this is antibody has been purposely designed to be used in ELISA (https:// www.sigmaaldrich.com/catalog/product/sigma/a3187?lang=en&region=GB) Flow cytometry: Each primary antibody was titrated in-house to determine optimal concentration. The use of the same lot number ensured consistency within the assay and over time.
• Anti-human CD14 eFluor450, clone HIB19-The manufacturer confirms on its website that this antibody has been purposely designed to be used in flow cytometry (https://www.thermofisher.com/antibody/product/CD14-Antibody-clone-61D3-Monoclonal/48-0149-42) • Anti-human CD19 eFluor450, clone 61D3-The manufacturer confirms on its website that this antibody has been purposely designed to be used in flow cytometry (https://www.thermofisher.com/antibody/product/CD14-Antibody-clone-61D3-Monoclonal/48-0149-42) • Anti-human CD3 AF700, clone UCHT1-The manufacturer confirms on its website that this antibody has been purposely designed to be used in flow cytometry and that it was verified by Relative expression to ensure that the antibody binds to the antigen stated (https://www.thermofisher.com/antibody/product/CD3-Antibody-clone-UCHT1-Monoclonal/56-0038-82) • Anti-human CD4 APC, clone RPA-T4-The manufacturer confirms on its website that this antibody has been purposely designed to be used in flow cytometry (https://www.thermofisher.com/antibody/product/CD4-Antibody-clone-RPA-T4-Monoclonal/17-0049-42) • Anti-human CD8a APC eFluor780, clone RPA-T8 -The manufacturer confirms on its website that this antibody has been purposely designed to be used in flow cytometry (https://www.thermofisher.com/antibody/product/CD8a-Antibody-clone-RPA-T8-Monoclonal/47-0088-42) • Anti-human IFN-y FITC, clone 4S.B3-The manufacturer confirms on its website that this antibody has been purposely designed to be used in flow cytometry and that it was verified by Relative expression to ensure that the antibody binds to the antigen stated nature research | reporting summary Note that full information on the approval of the study protocol must also be provided in the manuscript.

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Cells lines tested negative for mycoplasma contamination. None.
24 healthy adult UK residents, between 18 and 45years old, were recruited. Table 1 on this manuscript describes the baseline characteristics of the recruited participants.
Volunteers were recruited by use of advertisement formally approved by the ethics committees. Advertisement was distributed around the city of Oxford or posted on-line (University website and social media). Before any participant became enrolled, a formal in-person screening visit took place. During this visit all study procedures were explained, inclusion and exclusion criteria were assessed and informed consent was obtained. Volunteers were excluded from the study if they were concurrently involved in another trial. In order to check this, volunteers were asked to provide their National Insurance or Passport number (if they are not entitled to a NI number) and were registered on a national database of participants in clinical trials (www.tops.org.uk). There is a potential for self selection bias, but this is a recognised limitation of first-in-human studies where we are purposely selecting healthy adults rather than trying to obtain a sample that is representative of the general population. This is why we have planned further phase 1 / 2 studies with larger sample sizes.
This study was approved within the UK by the Medicines and Healthcare Products Regulatory Agency (MHRA reference 21584/0394/001-0001) and the South Central Oxford A Research Ethics Committee (REC reference 18/SC/0004).

NCT03590392
The study protocol is available with this publication as part of the supplementary material.
The study took place at a single site in the UK (Centre for Clinical Vaccinology and Tropical Medicine, University of Oxford). Participants' screenings and recruitment began in August 2018, with last participant's visit on 30th September 2019.
Study objectives and outcomes are described within the study protocol.
Primary Outcome Measures: The specific endpoints for safety and reactogenicity were actively and passively collected data on adverse events. The following parameters were assessed by: * Occurrence of solicited local reactogenicity signs and symptoms for 7 days following the vaccination. * Occurrence of solicited systemic reactogenicity signs and symptoms for 7 days following the vaccination. * Occurrence of unsolicited adverse events for 28 days following the vaccination. * Change from baseline for safety laboratory measures.