Background
Current malaria control strategies combine identification and elimination of vectors with prompt diagnosis and treatment of infected individuals in endemic populations. Various insecticides have been applied for the control of the mosquito vectors, and chemoprophylaxis is used to prevent blood stage infection associated with the clinical symptoms of malaria. These disease control measures are however being hampered by the resistance of parasites and mosquito vectors to drugs and insecticides respectively [
1]. Vaccines are an essential and cost-effective public health tool and it is believed that the development of anti-malarial vaccines would be an important addition to existing control strategies. The feasibility of developing a malaria vaccine is firstly suggested by the acquisition of partial clinical immunity following repeated exposure to parasites during natural transmission in malaria-endemic areas [
2,
3]. Secondly immunization with radiation-attenuated sporozoites has been shown to induce sterile protection against the sporozoite and liver stages of the parasite [
4,
5].
The malaria parasite has a complex life cycle and it is believed that an effective anti-malarial subunit vaccine may need to target antigens in multiple stages of the parasite. The
Plasmodium falciparum cell-traversal protein for ookinetes and sporozoites (CelTOS) is required for motility of the parasite in both the mosquito vector and the human host, and is required for successful malaria infections [
6]. Anti-CelTOS antibody responses in mice have been shown to inhibit sporozoite motility and invasion of hepatocytes
in vitro, and induced sterile protection in test animals [
7,
8]. Moreover, CelTOS peptides elicited proliferative and IFN-γ responses in
ex vivo ELISpot assays using peripheral blood mononuclear cells (PBMCs) from irradiated sporozoite-immunized volunteers [
9]. There are, however, no reports on the induction of naturally acquired cell-mediated immune responses to CelTOS in populations in malaria-endemic areas.
E
x vivo ELISpot assays have previously been shown to be capable of detecting recall IFN-γ responses from adults in Ghana using HLA-matched DR- or class I-restricted peptides derived from the circumsporozoite protein (CSP), thrombospondin-related adhesion protein (TRAP), liver stage antigen-1 (LSA1), liver stage antigen-3 (LSA3) and exported protein-1 (EXP1) [
10]. Moreover, since responses were generally low, in the same study non-HLA matched peptides from CSP and apical membrane antigen-1 (AMA1) were used to determine the reproducibility of
ex vivo ELISpot assays, and found that using positivity criteria defined as at least a two-fold difference comparing test samples and medium controls, and at least a difference of 10 spot forming cells (sfc) per million PBMC between test samples and medium controls, led to 60% reproducibility [
10]. The aim of this study was, therefore, to determine whether CelTOS peptides could induce IFN-γ recall responses in PBMCs from volunteers in Ghana using
ex vivo ELISpot assays, using the positivity criteria based on that study [
10]. Pools of peptides from CSP, TRAP, LSA1, AMA1 and merozoite surface protein-1 (MSP1) were concurrently used for PBMC stimulation. For each of these additional antigens, a single pool of peptides covering the entire antigen was used, rather than HLA-matched individual peptides, with the assumption that such pools contain multiple HLA-restricted epitopes that match the HLA of each subject. The results showed that with the exception of MSP1, CelTOS peptides recalled a similar frequency of positive responses as the other four antigens, suggesting that CelTOS may be a potentially important antigen for inclusion in a multi-antigen malaria vaccine.
Discussion
The development of an effective anti-malarial vaccine is an important public health priority as it will add to the currently available disease control tools. Subunit vaccine development requires the identification of conserved immunodominant antigens from multiple stages of the parasite in order to ensure broad protection and curtail disease transmission. CelTOS is a pre-erythrocytic malaria vaccine candidate antigen that has been shown to induce potent antibody and T cell responses and prevent the establishment of blood stage infection in mice [
6-
8]. CelTOS 15mer peptides also elicited HLA-restricted IFN-γ responses in
ex vivo ELISpot assays using PBMC from irradiated sporozoite-immunized volunteers [
9]. Moreover, CelTOS is a relatively conserved antigen and elicits cross-protective immune responses against heterologous challenge with
Plasmodium berghei [
6,
7]. CelTOS based vaccines have been shown to elicit both antibody and T cell responses in experimental animal models [
7,
8,
12,
13], and phase 1 clinical trials of a CelTOS vaccine known as FMP012 in malaria-naïve volunteers has just been completed (
https://clinicaltrials.gov/ct2/show/NCT01540474). However, very little is known about naturally induced immune responses to CelTOS in a population with exposure to
P. falciparum. A previous study with volunteers aged one to 30 years and living in a low malaria transmission area of southern Ghana showed very low levels of naturally induced CelTOS-specific antibodies [
14]. The aim of the current study was to investigate the induction of CelTOS-specific IFN-γ recall responses in PBMCs from Ghanaian adult volunteers in an area (Legon) with natural exposure to
P. falciparum parasites, which was previously confirmed by seropositivity to erythrocytic stage antigens [
10]. However, in that previous study, only a proportion of subjects had recall ELISpot activities to six malaria antigens that were tested, suggesting that cellular responses may be shorter-lived than antibody responses [
10].
Four pools of 15mer peptides that together span the entire amino acid sequence of CelTOS were tested against PBMCs from 35 study volunteers. Fourteen percent (14%) of volunteers made positive responses to the four CelTOS peptide pools, and all positive responses were to the first three peptide pools (CelTp1, CelTp2 and CelTp3) with no response to CelTp4 (Figures
1 and
2). By contrast, 49% of volunteers made positive responses to one or more of the six single peptide pools spanning the full length antigens (CelTTp, AMA1p, CSPp and TRAPp) or partial sequences (LSA1p and MSP1p), shown in Figures
3 and
4. Since one volunteer who was negative with the individual CelTOS pools, was positive to the single CelTTp pool, six/35 (17%) of volunteers were positive to CelTOS. This was not related to the viability of the PBMC, as all volunteers made positive responses to the Con A and CEF (Additional file
1), with the exception of volunteers v13, v16 and v26 whose cells at 100,000/well did not make responses to Con A, suggesting that PBMCs used for IFN-γ response assessment were viable. The magnitude of responses to CEF peptides in this study (Additional file
1) was generally similar to those in a previous study with volunteers from the same study area [
10]. The mean CEF response in PBMCs from the 29 volunteers with positive IFN-γ responses (1032 sfc/m) was however up to five times higher than the average response (216 sfc//m) measured in PBMCs from the two malaria-naïve volunteers (Additional file
2). In addition, anti-CelTOS IFN-γ responses observed in this study were several orders lower than the average response observed in a previous study with PBMCs from attenuated sporozoite-immunized volunteers [
9].
These results show that peptide pools from the N-terminal end of CelTOS (CelTp1, CelTp2, CelTp3) induced responses whereas the C-terminal peptide pool (CelTp4) did not (Figure
1). This suggests a greater prevalence of T cell epitopes in the N-terminal region of CelTOS. Preliminary predictions using the bioinformatic algorithm NetMHC [
15] identified more Class 1-restricted epitopes within CelTp1, CelTp2 and CelTp3 than CelTp4 [
9]. This is in agreement with the
ex vivo ELISpot results presented here and suggest that at least some of the responses observed in the current study are likely to be CD8+ T cell-specific. Despite the conserved nature of the
celtos gene, a limited number of single nucleotide polymorphisms (SNPs) have been mapped to the C-terminal region of the protein sequence that has been predicted to have immunodominant B cell epitopes [
16] and may thus be more involved in immune escape against CelTOS-specific antibody responses.
Ex vivo ELISpot analysis of mouse PBMCs showed a concentration of immunogenic T cell epitopes at the C terminal end of CelTOS, although murine HLA-restricted CD4+ and CD8+ epitopes were predicted by bioinformatics tools to be distributed over the entire antigen in the same study [
17]. It is possible that differences between murine and human HLA-restricted CelTOS epitopes may explain differences in ELISpot data between that study and the current study. However, on the basis of data from this and other human studies [
9], there is likely to be little effect of the limited polymorphism in CelTOS on anti-CelTOS T cell IFN-γ responses. Elucidation of the HLA restriction of observed responses by CD4+/CD8+ cell depletion ELISpot assays or by flow cytometric analysis and HLA-specific peptides will help determine the T cell subset specificity of observed responses and further establish the use of CelTOS as a malaria vaccine in genetically-diverse populations especially in Africa [
18].
In these studies the proportion of positive volunteers was greatest to the blood stage antigen MSP1 but statistically, only the difference between responders to MSP1 on the one hand and CSP and TRAP on the other hand was significant (Figure
4, Table
2). The proportions of positive volunteers to the other antigens were statistically similar. The overall magnitudes of responses were similar against all antigens tested; however, individual responses were highest against MSP1, LSA1 and TRAP in eight volunteers (Figure
3). These observations may be partially explained by the fact that blood stages persist for longer during malaria infection and thus induce responses in a greater proportion of volunteers (MSP1), while LSA1 contains more immunogenic epitopes than other malaria antigens [
19]. A number of studies in both naturally exposed individuals and naïve volunteers immunized with radiation-attenuated sporozoites have found numerous CD4+ and CD8+ T cell epitopes in MSP1, with most of these being limited especially to the 42 kDa fragment of protein [
20-
22]. However, the involvement of CD4+ or CD8+ T cells in these ELISpot activities was not determined in the current study.
In a previous study, short Class I-restricted and DR-restricted long peptides representing CSP, TRAP, and LSA1 were used as stimulants and these matched the HLA of volunteers in Ghana [
10]; in total 12/26 volunteers (46%) were positive, comparable with the total number 17/35 volunteers (49%) who were positive against CSP, TRAP and LSA1, in addition to AMA1 and MSP1 in this study (Figure
3). Thus pools of peptides spanning the entire length of malaria antigens were as effective in eliciting
ex vivo IFN-γ responses as HLA-matched single peptides. In that earlier study the highest frequency of responses was to LSA1 [
10] and MSP1 was not tested in that earlier study.
In summary, CelTOS-specific T cell responses were detected in five out of 35 study volunteers who have a history of exposure to
P. falciparum. The proportion of volunteers who responded to the CelTOS antigen was not significantly different from the proportions that responded to other established malaria vaccine candidate antigens. Immune responses to CelTOS may block the development of pre-erythrocytic parasites and have the potential to protect malaria endemic populations by reducing or preventing infection of liver cells by sporozoites, thereby reducing disease severity. Since natural transmission and immunization with irradiated sporozoites induce cellular IFN-γ responses to CelTOS, these findings support further evaluation of CelTOS as a pre-erythrocytic candidate antigen for inclusion in a potential multi-antigen vaccine. It has been shown previously that two vaccine candidate antigens, CSP and AMA1, combined in a DNA-prime, adenovirus-boost vaccine regimen induced complete protection that was primarily mediated by CD8+ T cell IFN-γ responses in 4/15 volunteers to malaria challenge [
23,
24]. It is possible that inclusion of other antigens such as CelTOS that also induce cellular IFN-γ responses may increase efficacy of this vaccine.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
Conceived and designed experiments – DA, KAK, SA, BG, MFO, TBK, DD, EV, MS. Performed the experiments – DA, KAK, HG, MB, JH, GB. Performed data analysis; KAK, EB, MRH, DD, MS. Contributed reagents/other resources – MS, EV, DD, BG, MFO. Wrote the paper – DA, KAK, HG, BG, TBK, MFO, EV, MRH, DD, KAK, MS. All authors read and approved the final manuscript.