T cell responses to the RTS,S/AS01(E) and RTS,S/AS02(D) malaria candidate vaccines administered according to different schedules to Ghanaian children

Abstract

Background: The Plasmodium falciparum pre-erythrocytic stage candidate vaccine RTS,S is being developed for protection of young children against malaria in sub-Saharan Africa. RTS,S formulated with the liposome based adjuvant AS01(E) or the oil-in-water based adjuvant AS02(D) induces P. falciparum circumsporozoite (CSP) antigen-specific antibody and T cell responses which have been associated with protection in the experimental malaria challenge model in adults.

Methods: This study was designed to evaluate the safety and immunogenicity induced over a 19 month period by three vaccination schedules (0,1-, 0,1,2- and 0,1,7-month) of RTS,S/AS01(E) and RTS,S/AS02(D) in children aged 5-17 months in two research centers in Ghana. Control Rabies vaccine using the 0,1,2-month schedule was used in one of two study sites.

Results: Whole blood antigen stimulation followed by intra-cellular cytokine staining showed RTS,S/AS01(E) induced CSP specific CD4 T cells producing IL-2, TNF-α, and IFN-γ. Higher T cell responses were induced by a 0,1,7-month immunization schedule as compared with a 0,1- or 0,1,2-month schedule. RTS,S/AS01(E) induced higher CD4 T cell responses as compared to RTS,S/AS02(D) when given on a 0,1,7-month schedule.

Conclusions: These findings support further Phase III evaluation of RTS,S/AS01(E). The role of immune effectors and immunization schedules on vaccine protection are currently under evaluation.

Trial registration: ClinicalTrials.gov NCT00360230.

Conflict of interest statement

Competing Interests: Johan Vekemans, Michel Janssens, Marc Lievens, Aurelie Olivier, Erik Jongert, Joe Cohen, and Philippe Moris are employees of GlaxoSmithKline Biologicals. Johan Vekemans and Joe Cohen own shares in GlaxoSmithKline. Joe Cohen was listed as inventor of patented malaria vaccines, including RTS,S. Barbara Savarese is an employee of MVI, which supports the development and testing of several malaria vaccines, and has financially supported this trial. Brian Greenwood reports having received grants by GSK for other studies. Patrice Dubois declares having received consultancy fees from GSK for this and other projects. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. None of the other authors report any potential conflicts of interest.

Figures

Figure 1. Schematic representation for evaluation of CSP specific T cell responses.

Triangles represent timing of vaccination for 0,1-, 0,1,2- and 0,1,7-month schedules (RTS,S/AS01E, RTS,S/AS02D or Rabies vaccine); arrows represent timing of blood sampling. In both research centers, each study group included 45 individuals.

Figure 2. Whole-blood intracellular cytokine detection by flow cytometry.

Whole-blood intracellular cytokine detection by flow cytometry was performed following overnight stimulation with medium (negative control), CSP and PHA (positive control). (A) CD4 or CD8 T cells were identified from a lymphocyte gate on an SSC-FSC plot. (B) IL-2+, TNF-α+, IFN-γ+, and CD40-ligand (not shown) CD4 T cells and CD8 T cells (not shown) were counted. The unstimulated sample (medium) shows background levels of cytokine production, while the stimulation with PHA (positive control) shows strong production of IL-2, TNF-α, or IFN-γ by CD4 T cells. The CSP stimulated illustrative sample from an RTS,S/AS01E vaccinated individual shows production of IL-2, TNF-α, and IFN-γ by CD4 T cells.

Figure 3. Frequency of CSP-specific CD4 T cells expressing at least IL-2, TNF-α or IFN-γ.

CSP-specific CD4 T cell responses in infants and children aged 5–17 months from Kintampo, vaccinated with RTS,S/AS01E or rabies vaccine according to a 0,1,2-month immunization schedule. Results are expressed as the median (with Q1 and Q3) number of CSP-specific CD4 T cells per 106 CD4 T cells. The number of subjects per group and percentage responders (defined as a response that was equal or greater than the geometric mean + 3 standard deviations (on the log 10 scale) of background stimulation) is indicated. P-values were calculated using the Wilcoxon Rank Sum test. *** P<0.001, ** P<0.01, * P<0.05.

Figure 4. Polyfunctional profiles of CSP-specific CD4 T cells one month post last immunization.

Polyfunctional profiles of CSP-specific CD4 T cells expressing any combination of immune markers among IL-2, TNF-α, IFN-γ, and CD40L in infants and children aged 5–17 months from Kintampo, vaccinated with RTS,S/AS01E or rabies vaccine according to a 0,1,2-month immunization schedule. Data are represented as background subtracted geometric mean number of CSP-specific CD4 T cells expressing any combination of IL-2, TNF-α, IFN-γ, and/or CD40L per 106 CD4 T cells, with 95% CI (A). The pie chart represents the proportion of CSP-specific CD4 T cells expressing 1, 2, 3 or 4 immune markers amongst IL-2, TNF-α, IFN-γ, and CD40L from RTS,S/AS01E recipients (B).