In this study we evaluated T cell responses to malarial antigens following vaccination with a recombinant blood stage malarial antigen (AMA1) formulated with aluminum hydroxide. Samples were obtained from two clinical trials, but the vaccination schedule, antigen formulation, and study populations were essentially identical. Presence of anti-AMA1 antibodies was tested and found to be negative pre-vaccination, and 2 weeks after receiving the second and third vaccination all volunteers responded to the polymorphic forms AMA1-FVO and AMA1-3D7, as determined by both IgG antibodies and antigen-driven cytokine secretion (Supplementary Table I
). Here, we focused on T cell responses which were analyzed using an approach that identifies multifunctional Th1 cells (producing IFN-γ, IL-2 and TNF-α and combinations of these cytokines) as a novel readout of vaccine-elicited T cell functionality for malarial antigens. Additionally we measured memory T cell subpopulations following each of three immunizations with the malaria antigen in a naïve population. To our knowledge, this is the first report detailing the development of malaria-associated multifunctional T cells in Ag-naïve humans.
Cellular immunity against malarial antigens has been evaluated in earlier studies, including proliferation and IFN-γ production by ELISPOT assay as measures of specific immune responses [12
]. Doubts have been expressed about the use of ex vivo IFN-γ ELISPOT data as the most appropriate method for evaluating protective immune responses, since the frequency of IFN-γ secreting cells usually wanes and may disappear within weeks [24
]. Some investigators have suggested that IFN-γ ELISPOT assays performed on PBMC cultured in vitro for 10 days may provide a better correlate for memory responses and protection against malaria [25
], but these assays only measure one cytokine. IL-2 is well known for its role in the induction of T-cell proliferation and recent studies have shown that the frequencies of cells that secrete a combination of IFN-γ and IL-2 were more closely associated than IFN-γ alone with immunogenicity and establishment of memory in malaria [26
], and other infectious diseases [27
]. Intracellular staining for IFN-γ-secreting cell frequencies in whole blood samples have been utilized in the evaluation of responses to Bacillus Calmette-Guerin (BCG) in studies of vaccines against tuberculosis [29
]. However, the studies performed limited analysis of cytokine secreting frequencies, focusing mainly on phenotyping of T cell subsets by surface marker expresssion. Other studies have concluded that the frequencies of T cells producing a combination of cytokines, chemokines and degranulation markers provide a better measurement of a robust immune response that may correlate with efficacy in vaccines against viral [18
], bacterial [31
] and parasitic infectious [16
]. Our goal in the present study was to characterize multifunctional T cell responses, with the hope of adapting the approach to whole blood samples in the future, which would be useful in field studies of malaria vaccines.
Aluminum hydroxide has been reported to induce predominantly Th2-biased T cell responses [33
] and, consistent with this, we found absolute frequencies of AMA1 stimulated IL-5 producing CD4 T cells to be about 20-fold higher than IFN-γ, or 3 fold more than IFN-γ and IL-2 (double-producing) cells. A recent report describing cellular immune responses to AMA1 vaccine formulated in Alhydrogel, showed increases in IFN-γ and IL-5 secretion, and a mean ratio of ELISPOT frequencies of IFN-γ/IL-5 of 1.16. In contrast, they reported more Th1-biased responses when the same antigen was formulated with Montanide ISA 720 or with AS02 as adjuvants [36
]. The significance of the expansion of IL-5 producing T cells following vaccination in humans is unknown, but in combination with IL-4, IL-5 may play an important role in generation and regulation of antibody responses.
Our results evaluating Th1 cytokine-producing cells after in vitro AMA1 stimulation demonstrated that triple-cytokine producers were very low in frequency, even after 3 vaccinations. Among double cytokine producers, the population with the highest frequency was the TNF-α+
double-positive subpopulation in both CD4 and CD8 T cell populations. Of the single cytokine producing cells, single TNF-α-producers had the highest baseline frequency within CD4 or CD8 T cell populations as previously reported [37
Although the absolute frequencies of total Th1 cytokine-producers showed no significant changes after immunization, the proportions of Th1 cytokine producing responders revealed an evolution that may represent differentiation or maturation of an immune response in both CD4 and CD8 T cells (). In CD4 T cells, the relative frequency of single TNFα-producing cells decreased whereas IL-2-producing cells increased strikingly from ~15% prior to vaccination (baseline) to ~80% by day 140. A transient change was detected after first vaccination where double positive TNF-α+
producing cells increased. The proportional frequency of this double positive cell population diminished, and may have later evolved to a dominant population of single IL-2-producers. Previous studies found a clear relationship between the role of IL-2 in cell proliferation and longevity of T cells and suggested that IL-2-only secreting cells are typical of central memory T cells that persist after antigen clearance [27
]. It has been further suggested that TNF-α/IL-2 producing T cells and single-IL-2-producing cells may constitute a “reservoir” population of memory T cells that can rapidly proliferate and differentiate to effector T cells upon re-exposure to infectious agent [39
]. Our findings are consistent with this model because of the high frequencies of these two populations and detection of memory markers.
Comparison of MFI values one week after the third vaccination suggested a hierarchy of cytokine secretion: triple cytokine-producing cells, albeit very low in absolute frequencies, produced more cytokine on a per cell basis than double-producers and the latter produced more cytokine than the single producers. This hierarchy is particularly consistent for IFN-γ production. In an animal model of leishmaniasis the iMFI values (a product of MFI by frequencies) calculated for IFN-γ correlated with protection [16
]. In our experiments, AMA1 vaccination resulted in a striking change in iMFI values for CD4 T cells. These findings suggest that analysis of the product of frequencies and MFIs may be a superior approach to evaluate T cell responses to malarial vaccines or natural infection, since frequencies alone give an incomplete picture of T-cell responses. Further studies with larger numbers of samples from either case-controlled study during natural infection, or following vaccination and challenge, will help us determine if such correlations exist.
Interestingly, our results indicated that in the CD8 T cell population, the proportion of IFN-γ producing cells increased 5–6 fold after the first vaccination and, as in the CD4 T cell population mentioned above, an expanded single-IL-2-producing population appeared later. Thus, CD8 T cells also underwent cytokine profile changes after AMA1 vaccination, although it has been widely accepted that blood stage antigens are almost exclusively presented to CD4 T cells. This finding may be explained by cross-presentation, a mechanism that involves uptake and processing of exogenous antigens within the major histocompatibility complex class I pathway [41
]. Of note, De Rosa and colleagues found that hepatitis B virus (HBV) immunization in humans may also induce significant CD8 responses, nearly identical in magnitude to CD4 responses [37
]. Future cellular studies on malaria will help to confirm if other blood stage malaria antigens are also capable of eliciting CD8 T cell responses.
We characterized the memory subpopulations based on CD45RO and CD62L expression. Comparison of the expression patterns before and after vaccination indicates that both central and effector memory responses are induced by AMA1 vaccination and that these memory subpopulations are evident as early as one week after the first vaccination. Most of our knowledge of the development of T cell memory is based on mouse models, particularly viral infection models inducing CD8 memory T cells [42
], while the maintenance and functional characteristics of memory CD4 T cells are less clear. The appearance of both CD4 Tcm
memory populations within 1 week of the first vaccination is consistent with a divergent model of memory cell generation as suggested previously [43
]. In a study using tetanus toxoid re-immunization with a technical approach similar to ours, Tem
could be identified as early as 5 days after re-immunization [44
], indicating that once a specific memory population has been established, rapid proliferation can occur upon to exposure to the target antigen. Malaria studies focusing on memory T cell populations will be important to elucidate the role of these different populations for protection, and to identify the determinants of longevity of memory responses.
In conclusion, we have demonstrated that AMA1/Alhydrogel® vaccination stimulates multifunctional cytokine producing cells in both CD4 and CD8 T cell populations. We have described the unique cytokine patterns induced by AMA1 vaccination, and also have shown that Ag-specific memory T cells, both Tem and Tcm, develop soon after immunization and can be detected in peripheral blood. These results demonstrate a successful new approach to the evaluation of cell-mediated immunogenicity of blood stage malaria vaccine candidates, which can be extended to other vaccine formulations with AMA1 and other parasite antigens. The detailed analysis of cellular immune responses based on cytokine profiles and memory cell development in this study enhances our ability to compare immune responses to specific malaria antigens qualitatively and quantitatively both in naïve individuals and in children and adults in malaria endemic areas.