Safety and immunogenicity of an AS03-adjuvanted plant-based SARS-CoV-2 vaccine in Adults with and without Comorbidities

CoVLP vaccine candidate and adjuvant

The CoVLP vaccine candidate consists of the full-length spike protein from SARS-CoV-2 (strain hCoV-19/USA/CA2/2020) incorporating the modifications R667G, R668S, R670S, K971P, and V972P. The modified spike protein was expressed in Nicotiana benthamiana by transient transfection, resulting in spontaneous trimer formation and CoVLP assembly and budding32. The CoVLPs were purified and formulated in phosphate-buffered saline (PBS) with polysorbate 80. The AS03 adjuvant is an oil-in-water emulsion containing DL-α-tocopherol and squalene, supplied by GSK. The placebo was PBS with polysorbate 80.

Vaccine preparation and injection

CoVLP was available in multi-dose vials (10 doses/vial) at 15 µg/mL and stored at 2–8 °C until shortly before use. The AS03 adjuvant was supplied in multi-dose vials (10 doses/vial). Prior to injection, 2.5 mL of CoVLP and 2.5 mL of AS03 were mixed to obtain 10 vaccine doses of 0.5 mL each. Each dose of the vaccine contained 3.75 µg of CoVLP formulated in PBS with polysorbate 80, 11.86 mg of DL-α-tocopherol and 10.69 mg of squalene. Once mixed, CoVLP+AS03 was stored at room temperature protected from light and had to be used within 6 h. All injections were administered intramuscularly in the deltoid using a 23-gauge needle of appropriate length based on body mass index (BMI). The first and second doses were administered contralaterally when possible.

Study design

The phase 2 portion of the study was a randomized, observer-blinded, placebo-controlled study with male and female participants. The study was approved by Advarra Central Institutional Review Boards (USA and Canada), IWK Health Centre Research Ethics Board, McGill University Health Centre Research Ethics Board, Comité d’Éthique de la Recherche du CHU de Québec – Université Laval as well as the Center for Biologics Evaluation and Research (CBER) of the U.S. Food and Drug Administration (FDA) and Biologic and Radiopharmaceutical Drugs Directorate (BRDD) of Health Canada and was carried out in accordance with the Declaration of Helsinki and the principles of Good Clinical Practices. Written informed consent was obtained from all study participants prior to any study procedure. Participants were offered modest compensation for their participation in this study (i.e.: time off work, displacement costs, etc.).

Participants were screened up to 14 days in advance of the first vaccine administration and must have demonstrated a satisfactory baseline medical assessment by history, general physical examination, hematologic, biochemic, and serologic analysis. Although a test for SARS-CoV-2 antibodies was performed at screening using a commercial ELISA that targets the nucleocapsid (N) protein (ElecSys: Roche Diagnostics), both seronegative and seropositive participants were enrolled.

For Healthy Adults, participants had to be 18–64 years of age. For Older Adults, participants had to be 65 years of age or older and to be non-institutionalized (e.g., not living in rehabilitation centers or old-age homes; living in an elderly community was acceptable). For Adults with Comorbidities, most frequent comorbidities were appetite and general nutritional disorders (obesity), allergic conditions, vascular hypertensive disorders, lipid metabolism disorders, glucose metabolism disorders (diabetes mellitus) and joint disorders (details in Supplementary Table 1). In this study, the Adults with Comorbidities group was older than the Healthy Adults group, had a different ethnic composition, and had a higher rate of baseline seropositivity (details in Table 1).

For both Healthy Adults and Older Adults, participants must have been in good general health with no clinically relevant abnormalities that could jeopardize subject safety or interfere with study assessments, as determined by medical history, physical examination, and vital signs, and have had a body mass index less than 30 kg/m2. Adults with Comorbidities included participants with one or more comorbid conditions that puts them at higher risk for severe COVID-19 such as obesity, hypertension, type 1 or type 2 diabetes, chronic obstructive pulmonary disease (COPD), cardiovascular diseases, chronic kidney diseases, or a compromised immune system (e.g., treatment-controlled HIV infection, organ transplant recipients, or patients receiving cancer chemotherapy). Female participants of childbearing potential must have had a negative pregnancy test result at screening and vaccination and used a highly effective method of contraception for one month prior to vaccination and at least one month after the last study vaccination. Exclusion criteria for Healthy Adults and Older Adults included i) any significant acute or chronic, uncontrolled medical or neuropsychiatric illness, ii) any chronic medical condition associated with elevated risk of severe outcome of COVID-19, iii) any confirmed or suspected current immunosuppressive condition or immunodeficiency, including cancer, HIV, hepatitis B or C infection, iv) current autoimmune disease, v) administration of any medication or treatment that could alter the vaccine immune response. In all three study populations, exclusion criteria also included vi) administration of any vaccine within 14 days prior to vaccination or planned administration of any vaccine up to Day 28 of the study, vii) administration of any other SARS-CoV-2 / COVID-19, or other experimental coronavirus vaccine at any time prior to or during the study, viii) history of virologically-confirmed COVID-19, ix) rash, dermatological condition, tattoos, muscle mass, or any other abnormalities at injection site that could interfere with injection site reaction rating, x) use of prophylactic medications (e.g., antihistamines [H1 receptor antagonists], nonsteroidal anti-inflammatory drugs [NSAIDs], systemic and topical glucocorticoids, non-opioid and opioid analgesics) within 24 h prior to the vaccination to prevent or pre-empt symptoms due to vaccination, xi) history of a serious allergic response to any of the constituents of CoVLP, including AS03, xii) history of documented anaphylactic reaction to plants or plant components (including tobacco, fruits and nuts), xiii) personal or family (first-degree relatives) history of narcolepsy, xiv) history of Guillain-Barré Syndrome. Sentinel participants (10 in each group) were first enrolled in Older Adults and Adults with Comorbidities groups, and unblinded safety data after each dose were reviewed by the Independent Data Monitoring Committee (IDMC). Enrollment into the Phase 2 portion of the study was closed on 25th March 2021.

The participants and the personnel collecting the safety information and working in testing laboratories remained blinded to treatment allocation. On D0, D21 and D42, serum and PBMC were processed for immune outcomes. All safety information was collected, and all laboratory procedures were carried out by study staff blinded to treatment allocation.

Primary and secondary objectives

The primary objectives of the Phase 2 portion of the study were to assess safety and tolerability and immunogenicity to CoVLP+AS03 at Study Day 0, Day 21 and Day 42, compared to placebo in Healthy Adults, Older Adults, and Adults with Comorbidities.

Primary safety outcomes were the occurrence(s) of i) immediate AEs within 30 min after each vaccination; ii) solicited local and systemic AEs up to 7 days after each vaccination; iii) unsolicited AEs, serious AEs (SAEs), AEs leading to withdrawal, AESIs, and deaths up to 21 days after each vaccination; iv) normal and abnormal urine, and hematological and biochemical values.

Primary immunogenicity outcomes were i) NAb titers measured using a pseudovirion neutralization assays and ii) IFN-γ and IL-4 ELISpot responses at 21 days after each dose of vaccine.

A secondary safety outcome was the occurrence(s) of SAEs, AEs leading to withdrawal, AESIs, and deaths from 22 days after the last vaccination up to the end of the study. Secondary immunogenicity outcomes were immune responses measured on study days 128, 201 and 386 The data collected up to the last time point of study day 386 will be released once study follow-up has been completed.

Safety assessments

In this manuscript, safety assessments are reported up to the cut-off date of April 28th, 2021. Both passive (diary) and active monitoring of safety signals were performed for the first 42 days of the study and continued throughout the study. Active monitoring included telephone contacts with participants one and eight days after each vaccination as well as a site visit on Day 3 after vaccination. Participants were required to return to the Investigator site on Days 128, 201, and 386 for safety follow-ups and immunogenicity assessments. In addition, study participants were contacted weekly to detect any symptoms that may be associated with COVID-19 and were instructed to report any changes in their health to the Investigator site.

Solicited AEs were assessed by the participants as Grade 1 to 4 (mild, moderate, severe, or potentially life-threatening) according to criteria described in the Protocol (see Supplementary Material)32. Per Protocol, all solicited AEs (local, systemic) were considered related events. Unsolicited adverse events were monitored for 21 days after each dose. SAEs, MAAEs, AEs leading to withdrawal and AESIs were collected throughout the study. Throughout the trial, an unblinded medical monitor (Syneos Health, Canada) was reviewing stopping rules from the trial and the Pharmacovigilance team at Medicago were reviewing AESIs (including VAED, anaphylaxis or severe allergic reactions, and potential immune-mediated disorders) and SAEs (including death). Methods for grading unsolicited AEs and trial-stopping rules are detailed in the Protocol. Unsolicited AEs were coded according to the terms used in the Medical Dictionary for Regulatory Activities (MedDRA), version 24.0.

Based on IDMC recommendations, the following event(s) could pause or halt the study for further review and assessment of the event(s): i) If any subject experienced an SAE after administration of the vaccine that was considered related to vaccine; ii) If 5% or more subjects who received the CoVLP formulation experienced the same or similar Grade 3 or higher solicited local AE or systemic AE, which began within 7 days after administration of the vaccine; or experienced the same or similar Grade 3 or higher unsolicited AE (including symptoms, signs or laboratory safety AEs) that was judged anything but unrelated to the vaccine; iii) If an important imbalance in unusual manifestations of COVID-19 or severity of COVID-19 symptoms was observed between the CoVLP and placebo groups.

SARS-CoV-2 pseudovirion and live virus neutralization assays, convalescent sera/plasma and WHO reference standard

The pseudovirion neutralization assay (Nexelis, Quebec, Canada) was based on a genetically modified Vesicular Stomatitis Virus (VSV) from which the glycoprotein G was removed, and a luciferase reporter introduced32. The modified VSV vector expresses full length SARS-CoV-2 S glycoprotein (NXL137-1 in POG2 containing 2019-nCOV Wuhan-Hu-1; Genebank: MN908947) from which the last nineteen amino acids of the cytoplasmic tail were removed (rVSVΔG-Luc-Spike ΔCT). Pseudovirions are mixed with sera of vaccinated individuals and the degree of neutralization is quantified using human ACE-2 expressing VERO cells and reduction in luciferase-based luminescence. For each sample, the neutralizing titer was defined as the reciprocal dilution corresponding to the 50% neutralization titer (NT50), when compared to the pseudovirion control without sera. Samples below cut-off were attributed a value of 5 (half the minimum required dilution).

Neutralizing antibody analysis was performed using a cell-based cytopathic effect assay (VisMederi, Sienna, Italy) based on ancestral SARS-CoV-2 virus (2019 nCOV ITALY/INMI1, provided by EVAg; Genebank: MT066156). For each sample, the neutralizing titer was defined as the reciprocal dilution corresponding to the NT50. Samples below cut-off were attributed a value of 5 (half the minimum required dilution). Full details of this assay are provided in Supplementary Methods.

For assessment of cross-reactivity against variants, the assay was conducted with live virus: Alpha (swab isolate 14484; mutations: N501Y, A570D, D614G, P678H, T716I, S982A, T572I, S735L, D69/70, D144Y), Beta (hCoV-19/Netherlands/NoordHolland_10159/2021), Gamma (human isolate PG_253 Clade Nexstrain 20 J/501Y.V3; Mutations: L18F, T20N, P26S, D138Y, R190T, K417T, E484K, N501Y, D614G, H655Y), Delta (sab isolate 31944, mutations: G142D, E156–158del, R158G, L452R, T478K, D614G, P681R, R582Q, D950N, V1061V), and Omicron (VMR_SARSCOV2_Omicron_C1, BA.1, Mutations: A67V, H69del, T95I, G142D, V143–145del, L212I, K417N, N440K, G446S, S477N, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K) variants.

Results were compared to sera/plasma from COVID-19 convalescent patients. These were collected from a total of 35 individuals with confirmed diagnosis. Time between the onset of the symptoms and sample collection varied between 27 and 105 days. Four sera samples were collected by Solomon Park (Burien, WA, USA) and 20 sera samples by Sanguine BioSciences (Sherman Oaks, CA, USA); all were from non-hospitalized individuals. Eleven plasma samples were collected from previously hospitalized patients at McGill University Health Centre. Disease severity was ranked as mild (COVID-19 symptoms without shortness of breath), moderate (shortness of breath reported), or severe (hospitalized). These samples were analyzed in parallel to clinical study samples, using the assays described above. Demographic characteristics have been previously described32.

To facilitate the comparability of results across different trials, the WHO International Standard for anti-SARS-CoV-2 immunoglobulin (human; NIBSC code: 20/136) was established to allow conversion of neutralization assay titers into international units (IU/mL). This standard consists of pooled plasma obtained from eleven individuals recovered from SARS-CoV-2 infection and with high NAb titers. Upon multiple assessments using the validated PNA assay, a conversion factor of 1.872 was established. Hence, the antibody titers presented throughout this manuscript could be expressed as IU/mL by dividing the NT50 by this factor. Similarly, a conversion factor of 0.91 could be applied to convert the live virus Wuhan strain titers to IU/mL.

Immunogenicity- interferon-γ and interleukin-4 ELISpot

PBMC samples from study participants were analyzed by IFN-γ or IL-4 ELISpots (Caprion, Quebec, Canada) using a pool of 15-mer peptides with 11 amino-acid overlaps from SARS-CoV-2 S protein (USA-CA2/2020, Genbank: MN994468.1, Genscript, purity >90%). Full details of the methodology are in Supplementary Methods.

Analysis populations and statistical analysis plan

Randomization was managed by Syneos Health (Canada) with Medicago oversight using Medidata Rave RTSM interactive randomization tool (2021.2.0, Medidata, USA). Statistical analysis and data presentation was conducted using SAS (SAS Institute, North Carolina) and Prism (GraphPad Software, San Diego).

The sample size of 753 participants made it possible to perform the initial evaluation of the vaccine immunogenicity and detect major differences in rates of AEs between groups. The sample size was not large enough to detect all types of, including less frequent or rare, AEs. The analyses of all immunogenicity endpoints were performed using randomized participants who received CoVLP+AS03 or placebo from the Intention-to-Treat population set. Immunogenicity was evaluated by humoral immune response (NAb assays) and cell-mediated immune response (ELISpot) induced in participants on D0, 21 and 42. To assess the humoral immune response, the GMT was calculated and compared between CoVLP+AS03 and placebo groups using an ANOVA on the log-transformed titers. The log transformation was used to meet the normal assumption for the ANOVA. At each time point, the GMT and corresponding 95CI of each treatment were obtained by exponential back-transformation of the least square mean. GMT were compared between study populations at D21 and D42 using an ANOVA. Comparison of D0 seronegative and seropositive values at D21 was conducted by unpaired t test of log-transformed values. Fisher’s exact test was used to compare seroconversion rate between the treatment groups. The 95CI for seroconversion was calculated using the exact Clopper-Pearson method. The specific Th1and Th2 responses along with the corresponding 95CI for the median induced on D0, D21 and D42 were measured by the number of cells expressing IFN-γ and IL-4 respectively, using ELISpot. The difference in IFN-γ and IL-4 response between treatment groups at each time point was compared using a non-parametric Wilcoxon Rank Sum Test. The difference in IFN-γ and IL-4 were also compared between study populations at D21 and D42 using a Kruskal-Wallis Test. Safety assessment are based on the Safety Analysis Set, i.e., all participants who received at least one dose of either the CoVLP+AS03 or placebo. Occurrence and incidence of safety events were reported for each treatment group. No formal hypothesis-testing analysis of AE incidence rates was performed and results were not corrected for multiple comparisons.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

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