Durable Human Memory T Cells Quantifiable by Cultured Enzyme-Linked Immunospot Assays Are Induced by Heterologous Prime Boost Immunization and Correlate with Protection against Malaria

Abstract

Immunological memory is a required component of protective antimalarial responses raised by T cell-inducing vaccines. The magnitude of ex vivo IFN-γ T cell responses is widely used to identify immunogenic vaccines although this response usually wanes and may disappear within weeks. However, protection in the field is likely to depend on durable central memory T cells that are not detected by this assay. To identify longer-lived memory T cells, PBMC from malaria-naive vaccinated volunteers who had received prime boost vaccinations with a combination of DNA and/or viral vectors encoding the multiepitope string-thrombospondin-related adhesion protein Ag were cultured in vitro with Ag for 10 days before the ELISPOT assay. Ex vivo T cell responses peaked at 7 days after the final immunization and declined substantially over 6 mo, but responses identified after T cell culture increased over the 6-mo period after the final immunization. Moreover, individual cultured ELISPOT responses at the day of challenge time point correlated significantly with degree of protection against malaria sporozoite challenge, whereas ex vivo responses did not, despite a correlation between the peak ex vivo response and magnitude of memory responses 6 mo later. This cultured assay identifies long-lasting protective T cell responses and therefore offers an attractive option for assessments of vaccine immunogenicity.

Multiple functions of human T cells generated by experimental malaria challenge

“…Our previous studies had shown the ratio of cultured:ex vivo responses to be in the order of 15-30, even shortly after vaccination, but here the ratio for the blood-stage Ag was 3.4 at day 28 and only 1.3 at day 90. The cultured response also fell significantly from the day 28 to the day 90 level, where a plateau might have been expected, as in our previous studies [21,32]. The concept that effector and memory T cells are differently regulated is highlighted by the finding that the ex vivo and cultured responses did not correlate either at day 28 or day 90.…”

“…The main assays used in this study were ex vivo and cultured ELISPOT, which measure effector and memory T-cell responses respectively, as extensively characterized by us and others [19][20][21]. A range of parasite density was observed in the individuals studied, reflecting the biological variability of infection despite a standardized infection procedure.…”

“…Cells were set up for ex vivo ELISPOT at 4 Â 10 6 mL À1 (50 mL/well), or in 1 mL cultures, in 24-well plates, at 1 Â 10 6 mL À1 with Ag, as optimized and described previously [21]. For the latter, on days 3 and 7, 0.5 mL of culture medium was removed and replaced with medium containing 100 U/mL IL-2 (Chiron), resulting in a final concentration of 50 U/mL.…”

“…Another CD4 1 phenotype, the Treg, can act by engagement of CTLA-4, or through secretion of inhibitory cytokines such as IL-10 or TGF-b, and their action is important for dampening excessive immune responses in malaria [3]. Functionally, T-cell responses can be divided into effector (or effector-memory) cells, which can be measured rapidly by overnight ELISPOT [19], and memory (or central memory) cells, which are revealed by a period of culture followed by ELISPOT, known as cultured ELISPOT [20,21]. Furthermore, expression of surface markers such as CCR7 and secretion of IL-2 are associated with proliferative memory T cells, which circulate in greater abundance once cognate Ag has disappeared from the system [22,23].…”

Alternating vector immunizations encoding pre‐erythrocytic malaria antigens enhance memory responses in a malaria endemic area

“…A study of naturally acquired immunity to CS protein demonstrated no correlation between cultured ELISPOT and ex vivo ELISPOT results [8]. The cells identified by cultured ELISPOT persist for at least 6 months after vaccination of naïve subjects, despite waning of cells detected by ex vivo ELISPOT [9]. The cells detected by cultured ELISPOT are here referred to as resting memory cells.…”