Induction of Plasmodium falciparum-specific CD4+ T cells and memory B cells in Gabonese children vaccinated with RTS,S/AS01(E) and RTS,S/AS02(D)

Abstract

The recombinant circumsporozoite protein (CS) based vaccine, RTS,S, confers protection against Plasmodium falciparum infection in controlled challenge trials and in field studies. The RTS,S recombinant antigen has been formulated with two adjuvant systems, AS01 and AS02, which have both been shown to induce strong specific antibody responses and CD4 T cell responses in adults. As infants and young children are particularly susceptible to malaria infection and constitute the main target population for a malaria vaccine, we have evaluated the induction of adaptive immune responses in young children living in malaria endemic regions following vaccination with RTS,S/AS01(E) and RTS,S/AS02(D). Our data show that a CS-specific memory B cell response is induced one month after the second and third vaccine dose and that CS-specific antibodies and memory B cells persist up to 12 months after the last vaccine injection. Both formulations also induced low but significant amounts of CS-specific IL-2(+) CD4(+) T cells one month after the second and third vaccine dose, upon short-term in vitro stimulation of whole blood cells with peptides covering the entire CS derived sequence in RTS,S. These results provide evidence that both RTS,S/AS01(E) and RTS,S/AS02(D) induced adaptive immune responses including antibodies, circulating memory B cells and CD4(+) T cells directed against P. falciparum CS protein.

Trial registration: ClinicalTrials.gov NCT00307021.

Figure 1. Flow of participants through the study.

Figure 2. Association between CS-specific antibody responses and the frequency of CS-specific IgG⁺ memory B cells.

The relationship between CS-specific antibody responses and the frequency of circulating CS-specific memory B cells was analysed at one month post dose 2 (month 2), one month post dose 3 (month 3) and one year post dose 3 (month 14). Data represent the CS-specific antibody level of each subject versus the corresponding frequency of CS-specific circulating memory B-cells for RTS,S/AS01_E and RTS,S AS02_D vaccine formulations. Number of responding subjects (n), Spearman correlation coefficient (R) and p-value are indicated.

Figure 3. Whole-blood intracellular cytokine detection by flow cytometry.

Whole-blood intracellular cytokine detection by flow cytometry was performed following overnight stimulation with SEB (positive control), the CS peptide pool or medium. (A) Cytokine production by CS-specific CD4⁺ T cells was determined by first dividing the CD3⁺ T cells in the lymphocyte gate into CD4⁺ or CD8⁺ T cells. (B) CD3⁺ CD4⁺ CD8⁻ T cells were analyzed with respect to the production of IL-2 and TNF-α. The unstimulated sample shows background levels of cytokine production, while stimulation with SEB shows strong production of IL-2 or TNF-α by CD4⁺ T cells. When restimulated with the CS peptide pool, the production of IL-2 by CD4⁺ T cells and low production of TNF-α was detected. (C) CD3⁺ CD4⁺ CD8⁻ T cells were analyzed for polyfunctional responses by simultaneous detection of IL-2 and TNF-α. While stimulation with SEB induces CD4⁺ T cells which produce the single cytokines and cells which produce both IL-2 and TNF-α, restimulation with the CS peptide pool induces mainly CD4⁺ T cells producing IL-2 only and a small fraction of cells is producing both IL-2 and TNF-α. The numbers in the quadrant gates of the plots denominates each distinct population based on their cytokine production. Samples from the same subject are shown, with responses at one month post dose 2. Results shown are representative of the range of responses seen with all subjects studied.

Figure 4. Induction of CD4⁺ T cell responses following vaccination with AS01 and AS02 based RTS,S formulations.

Frequency of CD4⁺ T cells expressing at least IL-2 were measured before vaccination, one month after the second vaccination (month 2), and one month (month 3) after the third vaccination (month 14). ICS data are represented for RTS,S/AS01_E and RTS,S AS02_D vaccine formulations, indicating Q1, Median, Q3. Significant differences against pre-values are indicated as *** p<0.001, ** p<0.01 and * p<0.05. Minimum and maximum frequencies of IL-2⁺ CD4⁺ T cell responses, the number of evaluated subjects and the responder rates are indicated.