Plasmodium falciparum-Specific Cellular Immune Responses after Immunization with the RTS,S/AS02D Candidate Malaria Vaccine in Infants Living in an Area of High Endemicity in Mozambique

Results from clinical trials in areas where malaria is endemic have shown that immunization with RTS,S/AS02A malaria vaccine candidate induces partial protection in adults and children and cellular effector and memory responses in adults. For the first time in a malaria vaccine trial, we sought to assess the cell-mediated immune responses to RTS,S antigen components in infants under 1 year of age participating in a clinical phase I/IIb trial of RTS,S/AS02D in Mozambique. Circumsporozoite protein (CSP)-specific responses were detected in approximately half of RTS,S-immunized infants and included gamma interferon (IFN-γ), interleukin-2 (IL-2), and combined IL-2/IL-4 responses. The median stimulation indices of cytokine-producing CD4⁺ and CD8⁺ cells were very low but significantly higher in RTS,S-immunized infants than in infants that received the comparator vaccine. Protection against subsequent malarial infection tended to be associated with a higher percentage of individuals with CSP-specific IL-2 in the supernatant (P = 0.053) and with higher CSP-specific IFN-γ-producing CD8⁺ T-cell responses (P = 0.07). These results report for the first time the detection of malaria-specific cellular immune responses after vaccination of infants less than 1 year of age and pave the way for future field studies of cellular immunity to malaria vaccine candidates.Malaria remains one of the major world heath problems affecting between 200 and 400 million people annually and causing 2 to 3 million deaths, mostly children and pregnant women living in sub-Saharan Africa (37). Infections by Plasmodium falciparum, one of the four species of plasmodia that affect humans, cause 80 to 90% of the malaria cases and are responsible for 95% of all malaria-associated deaths (14). Since most of the worldwide malaria burden is due to P. falciparum, efforts for prevention and eradication of malaria have focused on this parasite, and a P. falciparum-customized malaria vaccine is one of most promising tools (12, 25, 26).The most abundant and immunogenic antigen on the surface of Plasmodium sporozoites is the circumsporozoite protein (CSP), which is a target for vaccine development (9, 10, 17, 27). In vaccines based on irradiated sporozoites and CSP in human and mouse models, antibodies to circulating sporozoites, followed by cell-mediated responses to the protein after invasion of hepatocytes, have been described as crucial for the generation of protective responses (7, 11, 13, 28, 29).RTS,S is a subunit malaria vaccine candidate based on the CSP of P. falciparum that has been under study for many years. The chimeric vaccine contains a portion of the NANP-repeats, all four NVDP-repeats, and the complete carboxyl-terminal region of CSP suggested to be targets for humoral and cellular immunity, along with the amino-terminal region of HbsAg (HBS) (16). The malaria vaccine candidate RTS,S (GlaxoSmithKline, Rixensart, Belgium) formulated with the adjuvant system AS01 or AS02 has proven to confer partial protective immunity against malaria infection in malaria-naive adults (20, 21, 41), as well as in adults and infants in areas where malaria is endemic (2-6). Clinical safety, immunogenicity, and efficacy trials in infants and children have shown RTS,S/AS02 to be safe and protective and to induce high antibody titers (2, 4, 6, 34).Although the induction of a CSP-specific humoral response after RTS,S vaccination has been well described, the generation of cellular immune responses has not yet been addressed in infants or young children immunized with the RTS,S vaccine candidate. In adults, protection conferred by the RTS,S vaccine has been associated with acquisition of strong antibody and cellular responses to the CSP fragment of RTS,S (20, 22). Malaria naive volunteers immunized with RTS,S/AS02 frequently develop strong proliferative and IFN-γ-producing T-cell responses to peptides representing T-cell epitopes (Th2R and Th3R) present in the vaccine (22). A correlation between protection against experimental challenge and the CSP-specific production of IFN-γ by CD4⁺ and CD8⁺ T cells has been described in a limited number of individuals (42).Current efforts are under way to proceed to phase III clinical trials with the RTS,S vaccine, despite no currently identified immune correlates of protection for vaccination with RTS,S in infants or young children. The present study was integrated into a phaseI/IIb clinical trial of the RTS,S/AS02D candidate vaccine in infants in a rural area of malaria endemicity in Mozambique (4). We sought here to examine the cellular responses in infants vaccinated with RTS,S/AS02D and further the development of assays for use in malaria vaccine trials in infants and young children, the population most vulnerable to severe malaria.