Immunogenicity and safety of a quadrivalent plant-derived virus like particle influenza vaccine candidate-Two randomized Phase II clinical trials in 18 to 49 and ≥50 years old adults

Abstract

Background: New influenza vaccines eliciting more effective protection are needed, particularly for the elderly who paid a large and disproportional toll of hospitalization and dead during seasonal influenza epidemics. Low (≤15 μg/strain) doses of a new plant-derived virus-like-particle (VLP) vaccine candidate have been shown to induce humoral and cellular responses against both homologous and heterologous strains in healthy adults 18-64 years of age. The two clinical trials reported here addressed the safety and immunogenicity of higher doses (≥15 μg/strain) of quadrivalent VLP candidate vaccine on 18-49 years old and ≥50 years old subjects. We also investigated the impact of alum on the immunogenicity induced by lower doses of the vaccine candidate.

Method: In the first Phase II trial reported here (NCT02233816), 18-49 year old subjects received 15, 30 or 60 μg/strain of a hemagglutinin-bearing quadrivalent virus-like particle (QVLP) vaccine or placebo. In the second trial (NCT02236052), ≥50 years old subjects received QVLP as above or placebo with additional groups receiving 7.5 or 15 μg/strain with alum. Along with safety recording, the humoral and cell-mediated immune responses were analyzed.

Results: Local and systemic side-effects were similar to those reported previously. The QVLP vaccine induced significant homologous and heterologous antibody responses at the two higher doses, the addition of alum having little-to-no effect. Serologic outcomes tended to be lower in ≥50 years old subjects previously vaccinated. The candidate vaccine also consistently elicited both homologous and heterologous antigen-specific CD4+ T cells characterized by their production of interferon-gamma (IFN-γ), interleukine-2 (IL-2) and/or tumor-necrosis factor alpha (TNF-α) upon ex vivo antigenic restimulation.

Conclusion: Overall, the 30 μg dose produced the most consistent humoral and cellular responses in both 18-49 and ≥50 years old subjects, strongly supporting the clinical development of this candidate vaccine. That particular dose was chosen to test in the ongoing Phase III clinical trial.

Conflict of interest statement

BJW, NL, JC, and SP are paid employees of Medicago, and JFP and BYP are paid employees of Caprion Biosciences. Additionally, BJW has been a principal investigator of vaccine trials for several manufacturers, including Medicago Inc., for which his institution obtained research contracts. BJW has also received honoraria from several vaccine manufacturers for participation on Scientific Advisory Boards, including Medicago Inc. The Medicago’s VLP vaccine candidate is a product protected by patents owned by the company Medicago and the manuscript reports clinical results obtained with the candidate vaccine during a clinical trial authorized by United States of America and Canadian regulatory authorities. There are no other patents, products in development or marketed products associated with this research to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1

Disposition from screening to day 21 visit in (A) Adults (18-49y) and (B) Older Adults (≥50y).

Fig 2. Serum antibody response (HI titers) against the four homologous strains 21 days after vaccination in adults (18-49y).

(A) Geometric mean titers (GMT ± 95% CI), (B) Percent of seroprotection rate (SPR ± 95% CI), (C) Percent of seroconversion rate (SCR ± 95% CI) and (D) Geometric mean fold increase ratio (GMFR ± 95% CI). Histograms not connected by same letter are significantly different (P≤0.05, pair-wise comparison Tukey-Kramer test). The dotted line marks the values of the CHMP criteria.

Fig 3. Serum antibody response (HI titers) against the four homologous strains 21 days after vaccination in older adults (≥50y).

(A) Geometric mean titers (GMT ± 95% CI), (B) Percent of seroprotection rate (SPR ± 95% CI), (C) Percent of seroconversion rate (SCR ± 95% CI) and (D) Geometric mean fold increase ratio (GMFR ± 95% CI). Histograms not connected by same letter are significantly different (P≤0.05, pair-wise comparison Tukey-Kramer test). The gray zone marks the values of the CHMP criteria (the upper limit marks the values for adults ≥50y to 64y and the lower limit for adults ≥65y).

Fig 4. Impact of previous vaccination on serum antibody response (HI titers) against the four homologous influenza strains in older adults (≥50y).

(A) Percent of seroconversion rate (SCR ± 95% CI), (B) geometric mean fold increase ratio (GMFR ± 95% CI) between D0 and D21, (C) percent of seroprotection rate (SPR ± 95% CI) at D21, (D) geometric mean titer (GMT ± 95% CI) at D0, (E) geometric mean titer (GMT ± 95% CI) at D21. The gray zone marks the values of the CHMP criteria (the upper limit marks the values for adults ≥50y to 64y and the lower limit for adults ≥65y).

Fig 5. Serum antibody response (HI titers) against the four homologous strains 21 days after vaccination.

Geometric mean titer (GMT ± 95% CI) in (A) adult (18-49y) and (B) older adults (≥50y). Significant differences between vaccinated groups and Placebo are indicated (*P≤0.05, **P≤0.01, ***P≤0.001 pair-wise comparison Tukey-Kramer test). The dotted line or the gray zone (upper limit mark the values for adults ≥50y to 64y, lower limit for adults ≥65y) mark the values of the CHMP criteria.

Fig 6. CD4 T cell-mediated immune (% CD4 T cells) against homologous strains in adults (18-49y).

Median net changes (D21-D0) are represented. Plain symbols represent significant (P≤0.05, Wilcoxon matched-pairs signed rank) increase between D21 and D0 in opposition to open symbols indicating no significant increase of HA-specific CD4+ T cells ratio 21 days after vaccination. (A) Total response (i.e. the percentage of HA-specific CD4+ T cells secreting at least one of the three cytokines IFN-γ, TNF-α, IL-2) after ex vivo stimulation with the four VLP include in the vaccine. (B-E) Percentage of HA-specific CD4+ T cells secreting IFN-γ (Sum IFN-γ), TNF-α (Sum TNF-α), IL-2 (Sum IL-2) or at least two of these three cytokines (Sum poly) after ex vivo stimulation with (B) H1/Cal VLP, (C) H3/Vic VLP, (D) B/Bris VLP, (E) B/Mass VLP. Significant differences of the median net changes (D21-D0) are reported on each radar graphs (*P≤0.05, **P≤0.01, ***P≤0.01, Kruskal-Wallis test followed by Dunn’s multiple comparisons test).

Fig 7. CD4 T cell-mediated immune (% CD4 T cells) against homologous strains in older adults (≥50y).

Median net changes (D21-D0) are represented. Plain symbols represent significant (P≤0.05, Wilcoxon matched-pairs signed rank) increase between D21 and D0 in opposition to open symbols indicating no significant increase of HA-specific CD4+ T cells ratio 21 days after vaccination. (A) Total response (i.e. the percentage of HA-specific CD4+ T cells secreting at least one of the three cytokines IFN-γ, TNF-α, IL-2) after ex vivo stimulation with the four VLP include in the vaccine. (B-E) Percentage of HA-specific CD4+ T cells secreting IFN-γ (Sum IFN-γ), TNF-α (Sum TNF-α), IL-2 (Sum IL-2) or at least two of these three cytokines (Sum poly) after ex vivo stimulation with (B) H1/Cal VLP, (C) H3/Vic VLP, (D) B/Bris VLP, (E) B/Mass VLP. Significant differences of the median net changes (D21-D0) are reported on each radar graphs (*P≤0.05, **P≤0.01, ***P≤0.01, Kruskal-Wallis test followed by Dunn’s multiple comparisons test).

Fig 8. CD4 T cell-mediated immune (% CD4 T cells) against heterologous strains in adults (18-49y).

Median net changes (D21-D0) are represented. Plain symbols represent significant (P≤0.05, Wilcoxon matched-pairs signed rank) increase between D21 and D0 in opposition to open symbols indicating no significant increase of HA-specific CD4+ T cells ratio 21 days after vaccination. (A) Total response (i.e. the percentage of HA-specific CD4+ T cells secreting at least one of the three cytokines IFN-γ, TNF-α, IL-2) after ex vivo stimulation with the peptide pools of 15mer peptides overlapping by 11 amino acids spanning the complete HA sequences of four heterologous strains. (B-E) Percentage of HA-specific CD4+ T cells secreting IFN-γ (Sum IFN-γ), TNF-α (Sum TNF-α), IL-2 (Sum IL-2) or at least two of these three cytokines (Sum poly) after ex vivo stimulation with (B) H1/Bris peptide pool, (C) H3/Urug peptide pool, (D) B/Flo peptide pool, (E) B/Malaysia peptide pool. Significant differences of the median net changes (D21-D0) are reported on each radar graphs (*P≤0.05, **P≤0.01, ***P≤0.01, Kruskal-Wallis test followed by Dunn’s multiple comparisons test).

Fig 9. CD4 T cell-mediated immune (% CD4 T cells) against heterologous strains in older adults (≥50y).

Median net changes (D21-D0) are represented. Plain symbols represent significant (P≤0.05, Wilcoxon matched-pairs signed rank) increase between D21 and D0 in opposition to open symbols indicating no significant increase of HA-specific CD4+ T cells ratio 21 days after vaccination. (A) Total response (i.e. the percentage of HA-specific CD4+ T cells secreting at least one of the three cytokines IFN-γ, TNF-α, IL-2) after ex vivo stimulation with the peptide pools of 15mer peptides overlapping by 11 amino acids spanning the complete HA sequences of four heterologous strains. (B-E) Percentage of HA-specific CD4+ T cells secreting IFN-γ (Sum IFN-γ), TNF-α (Sum TNF-α), IL-2 (Sum IL-2) or at least two of these three cytokines (Sum poly) after ex vivo stimulation with (B) H1/Bris peptide pool, (C) H3/Urug peptide pool, (D) B/Flo peptide pool, (E) B/Malaysia peptide pool. Significant differences of the median net changes (D21-D0) are reported on each radar graphs (*P≤0.05, **P≤0.01, ***P≤0.01, Kruskal-Wallis test followed by Dunn’s multiple comparisons test).