Antibody responses to the RTS,S/AS01E vaccine and Plasmodium falciparum antigens after a booster dose within the phase 3 trial in Mozambique

Abstract

The RTS,S/AS01_E vaccine has shown consistent but partial vaccine efficacy in a pediatric phase 3 clinical trial using a 3-dose immunization schedule. A fourth-dose 18 months after the primary vaccination was shown to restore the waning efficacy. However, only total IgG against the immunodominant malaria vaccine epitope has been analyzed following the booster. To better characterize the magnitude, nature, and longevity of the immune response to the booster, we measured levels of total IgM, IgG, and IgG_1-4 subclasses against three constructs of the circumsporozoite protein (CSP) and the hepatitis B surface antigen (HBsAg, also present in RTS,S) by quantitative suspension array technology in 50 subjects in the phase 3 trial in Manhiça, Mozambique. To explore the impact of vaccination on naturally acquired immune responses, we measured antibodies to P. falciparum antigens not included in RTS,S. We found increased IgG, IgG1, IgG3 and IgG4, but not IgG2 nor IgM, levels against vaccine antigens 1 month after the fourth dose. Overall, antibody responses to the booster dose were lower than the initial peak response to primary immunization and children had higher IgG and IgG1 levels than infants. Higher anti-Rh5 IgG and IgG_1-4 levels were detected after the booster dose, suggesting that RTS,S partial protection could increase some blood stage antibody responses. Our work shows that the response to the RTS,S/AS01_E booster dose is different from the primary vaccine immune response and highlights the dynamic changes in subclass antibody patterns upon the vaccine booster and with acquisition of adaptive immunity to malaria.

Keywords: Adaptive immunity; Malaria; Vaccines.

Fig. 1. RTS,S/AS01_E booster and long-term immunogenicity against vaccine antigens: total IgG, IgG1-2 subclasses for CSP constructs and HBsAg at month (M) 20, 21, and 32 for RTS,S/AS01 vaccinees with (R3R) and without (R3C) booster, and comparator (C3C).

Boxplots with medians, interquartile ranges (IQR), upper whisker as the smallest between maximum × value and Q3 + 1.5*IQR, lower whisker as the largest between minimum × value and Q1 – 1.5*IQR, and log₁₀(geometric mean(MFI)) (diamond). Non-parametric tests were used to compare the booster response (M20 vs. M21) and the long-term immunogenicity (M21 vs. M32), as well as to compare the R3C and R3R groups at each timepoint. Only p-values < 0.05 after adjustment for multiple testing are shown. The y-axis is in logarithm 10 scale. R3R (green): three doses of RTS,S/AS01_E and a RTS,S/AS01_E booster. R3C (red): three doses of RTS,S/AS01_E and a comparator booster. C3C (blue): three doses of a comparator vaccine and a comparator booster.

Fig. 2. RTS,S/AS01_E booster and long-term immunogenicity against vaccine antigens: IgG3-4 subclasses and IgM for CSP constructs and HBsAg at month (M) 20, 21, and 32 for RTS,S/AS01 vaccinees with (R3R) and without (R3C) booster, and comparator (C3C).

Boxplots with medians, interquartile ranges (IQR), upper whisker as the smallest between maximum × value and Q3 + 1.5*IQR, lower whisker as the largest between minimum × value and Q1 – 1.5*IQR, and log₁₀(geometric mean(MFI)) (diamond). Non-parametric tests were used to compare the booster response (M20 vs. M21) and the long-term immunogenicity (M21 vs. M32), as well as to compare the R3C and R3R groups at each timepoint. Only p-values < 0.05 after adjustment for multiple testing are shown. The y-axis is in logarithm 10 scale. R3R (green): three doses of RTS,S/AS01_E and a RTS,S/AS01_E booster. R3C (red): three doses of RTS,S/AS01E and a comparator booster. C3C (blue): three doses of a comparator vaccine and a comparator booster.

Fig. 3. Antibody responses against vaccine antigens for months (M) 0, 3, 20, 21, and 32 for IgG, IgG1, and IgG2.

Boxplots with median, interquartile ranges (IQR), upper whisker as the smallest between maximum × value and Q3 + 1.5*IQR, and lower whisker as the largest between minimum × value and Q1 – 1.5*IQR. The y-axis is in logarithm 10 scale. Data from months 0 and 3 were obtained from a previous study in the same individuals, thus a batch effect might be present. R3R (green): three doses of RTS,S/AS01_E and a RTS,S/AS01_E booster at month 20. R3C (red): three doses of RTS,S/AS01_E and a comparator booster. C3C (blue): three doses of a comparator vaccine and a comparator booster.

Fig. 4. Antibody responses against vaccine antigens for months (M) 0, 3, 20, 21, and 32 for IgG3, IgG4 and IgM.

Boxplots with median, interquartile ranges (IQR), upper whisker as the smallest between maximum × value and Q3 + 1.5*IQR, and lower whisker as the largest between minimum × value and Q1 – 1.5*IQR. The y-axis is in logarithm 10 scale. Data from months 0 and 3 were obtained from a previous study in the same individuals [7], thus a batch effect might be present. R3R (green): three doses of RTS,S/AS01_E and a RTS,S/AS01_E booster at month 20. R3C (red): three doses of RTS,S/AS01_E and a comparator booster. C3C (blue): three doses of a comparator vaccine and a comparator booster.

Fig. 5. Immunogenicity for CSP FL stratified by previous clinical malaria: total IgG, IgG1-4 subclasses and IgM at month (M)20, 21, and 32 for RTS,S/AS01 vaccinees with (R3R) and without (R3C) booster, and comparator (C3C).

Stratified analysis by malaria cases before M20, subjects who presented with clinical malaria (M = blue) and subjects without malaria (NM = red). Boxplots with medians, interquartile ranges (IQR), upper whisker as the smallest between maximum × value and Q3 + 1.5*IQR, lower whisker as the largest between minimum × value and Q1 – 1.5*IQR, and log₁₀(geometric mean(MFI)) (diamond). Non-parametric tests were used to compare levels with or without clinical malaria (M vs. NM). p-values were adjusted for multiple comparisons, but none was significant. Only p-values < 0.05 before adjustment are shown. The y-axis is in logarithm 10 scale. R3R: three doses of RTS,S/AS01_E and a RTS,S/AS01_E booster. R3C: three doses of RTS,S/AS01_E and a comparator booster. C3C: three doses of a comparator vaccine and a comparator booster.

Fig. 6. Immunogenicity for CSP NANP stratified by previous clinical malaria: total IgG, IgG1-4 subclasses and IgM at month (M)20, 21, and 32 for RTS,S/AS01 vaccinees with (R3R) and without (R3C) booster, and comparator (C3C).

Stratified analysis by malaria cases before M20, subjects who presented with clinical malaria (M = blue) and subjects without malaria (NM = red). Boxplots with medians, interquartile ranges (IQR), upper whisker as the smallest between maximum × value and Q3 + 1.5*IQR, lower whisker as the largest between minimum × value and Q1 – 1.5*IQR, and log₁₀(geometric mean(MFI)) (diamond). Non-parametric tests were used to compare levels with or without clinical malaria (M vs. NM). p-values were adjusted for multiple comparisons, but none was significant. Only p-values < 0.05 before adjustment are shown. The y-axis is in logarithm 10 scale. R3R: three doses of RTS,S/AS01_E and a RTS,S/AS01_E booster. R3C: three doses of RTS,S/AS01_E and a comparator booster. C3C: three doses of a comparator vaccine and a comparator booster.

Fig. 7. Immunogenicity for CSP C-term stratified by previous clinical malaria: total IgG, IgG1-4 subclasses and IgM at month (M)20, 21, and 32 for RTS,S/AS01 vaccinees with (R3R) and without (R3C) booster, and comparator (C3C).

Stratified analysis by malaria cases before M20, subjects who presented with clinical malaria (M = blue) and subjects without malaria (NM = red). Boxplots with medians, interquartile ranges (IQR), upper whisker as the smallest between maximum × value and Q3 + 1.5*IQR, lower whisker as the largest between minimum × value and Q1 – 1.5*IQR, and log₁₀ (geometric mean(MFI)) (diamond). Non-parametric tests were used to compare levels with or without clinical malaria (M vs. NM). p-values were adjusted for multiple comparisons, but none was significant. Only p-values < 0.05 before adjustment are shown. The y-axis is in logarithm 10 scale. R3R: three doses of RTS,S/AS01_E and a RTS,S/AS01_E booster. R3C: three doses of RTS,S/AS01_E and a comparator booster. C3C: three doses of a comparator vaccine and a comparator booster.

Fig. 8. Immunogenicity stratified by clinical malaria after M21: total IgG, IgG1-4 subclasses and IgM for CSP FL at month (M) 20, 21, and 32 for RTS,S/AS01 vaccinees with (R3R) and without (R3C) booster, and comparator (C3C).

Stratified analysis by malaria after M21, subjects who presented with clinical malaria (M = blue) and subjects without malaria (NM = red). Subjects who presented with clinical malaria before M20 are represented with green and orange squares. Boxplots with medians, interquartile ranges (IQR), upper whisker as the smallest between maximum × value and Q3 + 1.5*IQR, lower whisker as the largest between minimum × value and Q1 – 1.5*IQR, and log₁₀ (geometric mean(MFI)) (diamond). Non-parametric tests were used to compare levels with or without clinical malaria (NM vs. M). p-values were adjusted for multiple comparisons, but none was significant. Only p-values < 0.05 before adjustment are shown. The y-axis is in logarithm 10 scale. R3R: three doses of RTS,S/AS01_E and a RTS,S/AS01_E booster. R3C: three doses of RTS,S/AS01_E and a comparator booster. C3C: three doses of a comparator vaccine and a comparator booster.

Fig. 9. Immunogenicity stratified by clinical malaria after M21: total IgG, IgG1-4 subclasses and IgM for CSP NANP at month (M) 20, 21, and 32 for RTS,S/AS01 vaccinees with (R3R) and without (R3C) booster, and comparator (C3C).

Stratified analysis by malaria after M21, subjects who presented with clinical malaria (M = blue) and subjects without malaria (NM = red). Subjects who presented with clinical malaria before M20 are represented with green and orange squares. Boxplots with medians, interquartile ranges (IQR), upper whisker as the smallest between maximum × value and Q3 + 1.5*IQR, lower whisker as the largest between minimum × value and Q1 – 1.5*IQR, and log₁₀(geometric mean(MFI)) (diamond). Non-parametric tests were used to compare levels with or without clinical malaria (NM vs. M). p-values were adjusted for multiple comparisons, but none was significant. Only p-values < 0.05 before adjustment are shown. The y-axis is in logarithm 10 scale. R3R: three doses of RTS,S/AS01_E and a RTS,S/AS01_E booster. R3C: three doses of RTS,S/AS01_E and a comparator booster. C3C: three doses of a comparator vaccine and a comparator booster.

Fig. 10. Immunogenicity stratified by clinical malaria after M21: total IgG, IgG1-4 subclasses and IgM for CSP C-term at month (M) 20, 21, and 32 for RTS,S/AS01 vaccinees with (R3R) and without (R3C) booster, and comparator (C3C).

Stratified analysis by malaria after M21, subjects who presented with clinical malaria (M = blue) and subjects without malaria (NM = red). Subjects who presented with clinical malaria before M20 are represented with green and orange squares. Boxplots with medians, interquartile ranges (IQR), upper whisker as the smallest between maximum × value and Q3 + 1.5*IQR, lower whisker as the largest between minimum × value and Q1 – 1.5*IQR, and log₁₀(geometric mean(MFI)) (diamond). Non-parametric tests were used to compare levels with or without clinical malaria (NM vs. M). p-values were adjusted for multiple comparisons, but none was significant. Only p-values < 0.05 before adjustment are shown. The y-axis is in logarithm 10 scale. R3R: three doses of RTS,S/AS01_E and a RTS,S/AS01_E booster. R3C: three doses of RTS,S/AS01_E and a comparator booster. C3C: three doses of a comparator vaccine and a comparator booster.

Fig. 11. Immunogenicity stratified by clinical malaria after M21: total IgG, IgG1-4 subclasses and IgM for HBsAg at month (M) 20, 21, and 32 for RTS,S/AS01 vaccinees with (R3R) and without (R3C) booster, and comparator (C3C).

Stratified analysis by malaria after M21, subjects who presented with clinical malaria (M = blue) and subjects without malaria (NM = red). Subjects who presented with clinical malaria before M20 are represented with green and orange squares. Boxplots with medians, interquartile ranges (IQR), upper whisker as the smallest between maximum × value and Q3 + 1.5*IQR, lower whisker as the largest between minimum × value and Q1 – 1.5*IQR, and log₁₀(geometric mean(MFI)) (diamond). Non-parametric tests were used to compare levels with or without clinical malaria (NM vs. M). p-values were adjusted for multiple comparisons, but none was significant. Only p-values < 0.05 before adjustment are shown. The y-axis is in logarithm 10 scale. R3R: three doses of RTS,S/AS01_E and a RTS,S/AS01_E booster. R3C: three doses of RTS,S/AS01_E and a comparator booster. C3C: three doses of a comparator vaccine and a comparator booster.

Fig. 12. RTS,S/AS01_E booster and long-term immunogenicity against the blood stage antigen Rh5: total IgG, IgG1-4 subclasses and IgM at month (M) 20, 21, and 32 for RTS,S/AS01 vaccinees with (R3R) and without (R3C) booster, and comparator (C3C).

Boxplots with medians, interquartile ranges (IQR), upper whisker as the smallest between maximum × value and Q3 + 1.5*IQR, lower whisker as the largest between minimum × value and Q1 – 1.5*IQR, and log₁₀(geometric mean(MFI)) (diamond). Non-parametric tests were used to compare the booster response (M20 vs. M21) and the long-term immunogenicity (M21 vs. M32), as well as to compare the R3C and R3R groups at each timepoint. Only p-values < 0.05 after adjustment for multiple testing are shown. The y-axis is in logarithm 10 scale. R3R (green): three doses of RTS,S/AS01_E and a RTS,S/AS01_E booster. R3C (red): three doses of RTS,S/AS01_E and a comparator booster. C3C (blue): three doses of a comparator vaccine and a comparator booster.

Fig. 13. Antibody responses against the blood stage antigen Rh5 for months (M) 0, 3, 20, 21, and 32 for IgG, IgG1, IgG2, IgG3, IgG4 and IgM.

Boxplots with median, interquartile ranges (IQR), upper whisker as the smallest between maximum × value and Q3 + 1.5*IQR and lower whisker as the largest between minimum × value and Q1 – 1.5*IQR. The y-axis is in logarithm 10 scale. Data from months 0 and 3 were obtained from a previous study in the same individuals [7], thus a batch effect might be present. R3R (green): three doses of RTS,S/AS01_E and a RTS,S/AS01_E booster at month 20. R3C (red): three doses of RTS,S/AS01_E and a comparator booster. C3C (blue): three doses of a comparator vaccine and a comparator booster.