GRAd-COV2 vaccine provides potent and durable humoral and cellular immunity to SARS-CoV-2 in randomized placebo-controlled phase 2 trial

Abstract

The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic and heterologous immunization approaches implemented worldwide for booster doses call for diversified vaccine portfolios. GRAd-COV2 is a gorilla adenovirus-based COVID-19 vaccine candidate encoding prefusion-stabilized spike. The safety and immunogenicity of GRAd-COV2 is evaluated in a dose- and regimen-finding phase 2 trial (COVITAR study, ClinicalTrials.gov: NCT04791423) whereby 917 eligible participants are randomized to receive a single intramuscular GRAd-COV2 administration followed by placebo, or two vaccine injections, or two doses of placebo, spaced over 3 weeks. Here, we report that GRAd-COV2 is well tolerated and induces robust immune responses after a single immunization; a second administration increases binding and neutralizing antibody titers. Potent, variant of concern (VOC) cross-reactive spike-specific T cell response peaks after the first dose and is characterized by high frequencies of CD8s. T cells maintain immediate effector functions and high proliferative potential over time. Thus, GRAd vector is a valuable platform for genetic vaccine development, especially when robust CD8 response is needed.

Keywords: CD4; CD8; COVID-19; Sars-CoV-2 vaccine; T cell response; immunological memory; neutralizing antibodies; phase 2 clinical trial; safety; simian adenoviral vector.

Graphical abstract

Figure 1

Trial profile All participants were followed up with in blind condition up to day 57 after the first dose; afterward, the randomization code was opened to allow participants assigned to the placebo group to have access to the vaccination campaign. The CONSORT diagram reports the number of participants in the full analysis set (FAS) at each time point up to day 180 and the corresponding number of participants included in the immunological analysis set (IAS). FAS included all randomized participants who received the dose of the investigational medicinal product (IMP), irrespective of their protocol adherence and continued participation in the study; IAS included all participants in the safety analysis set who had immune response assessments and no protocol deviations (or up to the time point before the deviation occurred) judged to have a potential interference with the generation or interpretation of an immune response (SARS-CoV-2 infection or commercial COVID-19 vaccination).

Figure 2

Frequency of participants with solicited systemic and local adverse events and severity Data are percentage of participants. (A) Frequency of participants with solicited systemic and local adverse events (AEs) within 7 days after the first dose. (B) Frequency of participants with solicited systemic and local AEs within 7 days after the second dose. Only events with a frequency ≥1% are reported. (C) Frequency of participants with solicited local and systemic AEs (any) within 7 days after the first and second dose according to the age category (18–65 years; >65 years). (D) Most frequent (>1%) AEs (solicited and unsolicited) within 28 days after any dose of vaccination. Severity was assessed for AEs according to toxicity grading scales modified and abridged from the US FDA guidance. No grade 4 AEs were observed.

Figure 3

Spike/RBD binding and SARS-CoV2 neutralizing antibody kinetics in GRAd-COV2-vaccinated and placebo arms (A–D) The magnitude and kinetics of antibodies binding to full-length trimeric spike (A) or RBD (B) and of live SARS-CoV-2 neutralizing antibodies, expressed as 50% (C) or 80% neutralizing titer (D), following GRAd-COV2 or placebo administration are reported over 6 months of follow up. Datapoints are the geometric mean (GM) and 95% confidence interval (CI) at each study visit for each study arm. For binding antibodies (A and B), data are expressed as arbitrary units (AU)/mL, as per assay manufacturer datasheet. For neutralizing antibody titers (C and D), data are expressed as NT₅₀ or NT₈₀, or the reciprocal of serum dilutions showing 50% or 80% infection reduction. Arrowheads below the x axes indicate vaccination. Statistical analysis of variance, as described in STAR Methods, is displayed only for comparison between SD and RD vaccine arms; the difference between placebo and both vaccine arms was highly significant (p ≤ 0.0001) at all post-vaccination visits. (E and F) Neutralizing titers at day 36 visit in a subset of 100 subjects in SD or RD vaccine study arms, measured by SARS-CoV-2 pseudoparticle neutralization assay (PNA) based on VSV pseudotyped with spike from Wuhan (filled symbols) or Delta (open symbols) strains. In (E), data are expressed as 50% neutralization titer (see above for definition), while in (F) and for the Wuhan strain only as appropriate, data are converted in WHO international units (IU)/mL. Each symbol corresponds to one serum sample, and horizontal line and error bars represent GM and 95% CI, respectively. Two-tailed Mann-Whitney test was used, and the only significant difference is shown in (E). Dashed lines indicate assay LOD. In all panels, gray symbols/lines indicate placebo arm, while red and blue symbols/lines indicate SD and RD GRAd-COV2 arms, respectively.

Figure 4

Spike-specific T cell response after GRAd-COV2 vaccination PBMCs were isolated and cryopreserved for the analysis of T cell responses from a subset of 54 volunteers, 21 enrolled in placebo (PL), 17 in SD, and 16 in RD arms. (A) Total T cell response to SARS-CoV-2 spike at days 22 (post-dose 1-PD1) and 36 (post-dose 2-PD2), evaluated by IFNγ ELISpot and expressed as IFNγ spot-forming cells (SFC) per million PBMCs. (B) Breadth of response to spike: response to DMSO (negative control: gray symbols) and peptide pools covering the S1a (pink symbols), S1b (green symbols), S2a (purple symbols), and S2b (violet symbols) portions of spike, evaluated in ELISpot at day 22. (C) Cross-reactivity of the T cell response to variants of concern: total spike response to Wuhan and Delta or Wuhan and Omicron variants, evaluated in distinct ELISpot assays using day 22 PBMCs from all GRAd-COV2-vaccinated subjects. The response on each variant in an individual volunteer is connected by a line, and bars are set at GM. (D) IFNγ (Th1) and IL-5 (Th2) production upon spike peptide pool stimulation, evaluated at the day 36 visit in eight subjects per vaccine arm and two PL recipients by two-color ELISpot. (E and F) Multiparametric flow cytometry analysis of CD4 and CD8 T cells responses at day 36 in all GRAd-COV2 and eight PL recipients. Total spike response (E) and breadth (F) of response on S1 and S2 spike domains. Data are expressed as the percentage of CD4 and CD8 expressing any combination of the analyzed functions (IFNγ, TNF-α, IL-2, or CD107a) within the CD69⁺ fraction in response to spike antigen stimulation. Pie charts (base: median) representing the functional profile of spike-specific CD4 and CD8 are shown in (E) and are better described in Figure S7. In all panels, red circles represent SD arm subjects, blue circles represent RD arm subjects, gray circles represent PL arm subjects, and black horizontal lines indicate GM.

Figure 5

Long-term T and B memory response in the PBMC substudy (A) Kinetics of T cell response to SARS-CoV-2 spike at days 22 (post-dose 1-PD1), 36 (post-dose 2-PD2), and 180, evaluated by IFNγ ELISpot and expressed as IFNγ spot-forming cells (SFC) per million PBMCs. (B) Proliferative CD4 and CD8 T cell responses (percentage of CD4 or CD8 CellTrace low) following 5 day incubation with spike peptide pools, assessed in PBMCs collected at day 180 (day 36 for the four PL arm subjects). (C) Quantification of spike-specific (Spike++ as defined in STAR Methods) memory B cell (MBC) percentages in all 54 PBMC substudy subjects at day 36 and 180 visits. Data were analysed with two-tailed, paired Wilcoxon test, and only significant differences are shown. In all panels, gray symbols/lines indicate PL arm, while red and blue symbols/lines indicate SD and RD GRAd-COV2 arms, respectively. Open red symbols and pink shaded areas indicate volunteers in the SD cohort that received an approved COVID-19 vaccine (defined SD+vax) between the day 57 and 180 visits. Horizontal black lines are set at GM.