Skip to main content
NCBI home page
Molecular Therapy logoLink to Molecular Therapy
. 2014 Jul 29;22(11):1992–2003. doi: 10.1038/mt.2014.109

Translating the Immunogenicity of Prime-boost Immunization With ChAd63 and MVA ME-TRAP From Malaria Naive to Malaria-endemic Populations

Domtila Kimani 1, Ya Jankey Jagne 2, Momodou Cox 2, Eva Kimani 1, Carly M Bliss 3, Evelyn Gitau 1, Caroline Ogwang 1, Muhammed O Afolabi 2, Georgina Bowyer 3, Katharine A Collins 3, Nick Edwards 3, Susanne H Hodgson 4, Christopher J A Duncan 4, Alexandra J Spencer 3, Miguel G Knight 1, Abdoulie Drammeh 2, Nicholas A Anagnostou 4, Eleanor Berrie 5, Sarah Moyle 5, Sarah C Gilbert 3, Peninah Soipei 1, Joseph Okebe 2, Stefano Colloca 6, Riccardo Cortese 6,7, Nicola K Viebig 8, Rachel Roberts 4, Alison M Lawrie 4, Alfredo Nicosia 6,7,9, Egeruan B Imoukhuede 8, Philip Bejon 1, Roma Chilengi 1, Kalifa Bojang 2, Katie L Flanagan 2,10, Adrian V S Hill 3,4, Britta C Urban 1,11, Katie J Ewer 3,*
PMCID: PMC4188556  EMSID: EMS59084  PMID: 24930599

Abstract

To induce a deployable level of efficacy, a successful malaria vaccine would likely benefit from both potent cellular and humoral immunity. These requirements are met by a heterologous prime-boost immunization strategy employing a chimpanzee adenovirus vector followed by modified vaccinia Ankara (MVA), both encoding the pre-erythrocytic malaria antigen ME-thrombospondin-related adhesive protein (TRAP), with high immunogenicity and significant efficacy in UK adults. We undertook two phase 1b open-label studies in adults in Kenya and The Gambia in areas of similar seasonal malaria transmission dynamics and have previously reported safety and basic immunogenicity data. We now report flow cytometry and additional interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) data characterizing pre-existing and induced cellular immunity as well as anti-TRAP IgG responses. T-cell responses induced by vaccination averaged 1,254 spot-forming cells (SFC) per million peripheral blood mononuclear cells (PBMC) across both trials and flow cytometry revealed cytokine production from both CD4+ and CD8+ T cells with the frequency of CD8+ IFN-γ-secreting monofunctional T cells (previously shown to associate with vaccine efficacy) particularly high in Kenyan adults. Immunization with ChAd63 and MVA ME-TRAP induced strong cellular and humoral immune responses in adults living in two malaria-endemic regions of Africa. This prime-boost approach targeting the pre-erythrocytic stage of the malaria life-cycle is now being assessed for efficacy in a target population.

Introduction

Approximately 1.2 billion people worldwide are at high risk of malaria and there were an estimated 207 million cases of malaria in 2012, with 90% occurring in sub-Saharan Africa and 57% of the African population living in areas of moderate to high transmission intensity.1,2 Malaria caused by Plasmodium falciparum remains one of the leading causes of death in children under 5 years of age in sub-Saharan Africa, despite the deployment of multiple control strategies such as the distribution of insecticide-treated nets, indoor residual spraying, and intermittent preventive treatment.3 An effective infant vaccine for malaria could substantially reduce the burden of disease and levels of malaria mortality and morbidity. The 2013 Malaria Vaccine Funders Group malaria vaccine roadmap has a landmark goal of producing a first-generation malaria vaccine by 2015 with greater than 50% efficacy against severe disease with greater than 1 year's duration.4 The current leading malaria vaccine candidate, RTS,S, is undergoing phase 3 trials across Africa in some 16,000 infants, however despite inducing sterile efficacy in a high proportion of malaria-naive adult volunteers,5 observed efficacy of vaccination with RTS,S/AS02 was only 30% in the target age group of 6–12-week-old infants and 50% among 5–17 month olds in malaria-endemic regions.6 Immune responses induced by vaccination with RTS,S include both humoral- and T-cell responses, however the cellular immune response is exclusively CD4+ T-cell-mediated and relatively modest.7,8 Studies of protective efficacy in rodent models repeatedly show that high frequencies of CD8+ T cells are essential for sterile protection9; this and evidence from animal models10,11,12 and studies in humans of vaccination with attenuated sporozoites,13 suggest that a successful malaria vaccine will benefit from both a humoral and cellular component. Therefore, it is highly likely that an additional component capable of generating high frequencies of CD8+ T cells will be beneficial to complement the protection afforded by RTS,S or any other antibody-inducing malaria vaccine.14

The Jenner Institute at Oxford University has been developing a program of viral vectored vaccines against infectious diseases including malaria for the past 14 years, the most promising of which utilizes the full length pre-erythrocytic antigen thrombospondin-related adhesive protein (TRAP) fused to a multi-epitope (ME) string of known T-cell epitopes.15 In previous studies with the ME-TRAP antigen, initial results in malaria naive volunteers in the United Kingdom were promising with strategies such as priming with a fowlpox vector (FP9) and boosting with modified vaccinia Ankara (MVA),16 however results of subsequent immunogenicity studies in target populations in Africa were disappointing with immune responses detected at levels only a small fraction of those in naive adult subjects.17,18 In consecutive studies, responses in adult UK volunteers averaged 475 spot-forming cells (SFC) per million peripheral blood mononuclear cells (PBMC) compared to 389 SFC/106 PBMC in coastal Kenya, which fell to 105 SFC/106 PBMC in Kenyan children aged 1–6 years demonstrating the difficulty in translating immunogenicity outcomes between populations.16,19,20

More recently, very potent cellular immune responses to ME-TRAP have been elicited by replacing the poxvirus priming vector with a chimpanzee adenovirus (ChAd63).21,22,23 The advantages of using chimpanzee adenoviruses rather than human serotypes include an excellent safety profile and enhanced immunogenicity as a consequence of lower levels of pre-existing neutralizing antibodies against the vector in the target populations for vaccination. A phase 1 safety and dose-escalation study demonstrated a good safety profile for ChAd63 ME-TRAP, and when combined with MVA ME-TRAP in a heterologous prime-boost approach induced T-cell frequencies of more than 2,000 SFC per million PBMC in adult UK volunteers.21

Between May 2010 and May 2011, we undertook two phase 1b trials of heterologous prime-boost with ChAd63-MVA ME-TRAP to study the effect of vaccine dose and route of administration on safety and immunogenicity profiles in Kenyan and Gambian adults. The trials were carried out in areas of similar malaria transmission characteristics. Volunteers received a priming vaccination with the simian adenovirus ChAd63, followed by a boosting dose of MVA 8 weeks later, with both vectors encoding ME-TRAP antigen. Previous immunogenicity and efficacy studies with this regimen in UK volunteers showed a good safety profile, very potent T-cell induction, and protection against experimental malaria in a controlled human malaria infection (CHMI) model.21 Efficacy was observed both through sterilizing immunity in 21% of recipients and a significant delay to patent parasitemia as determined by blood film microscopy in a further 34% of vaccinees, thus giving some degree of efficacy in more than half of vaccinees.24

Primary outcomes for these two phase 1b studies in malaria-endemic areas and the UK CHMI study reported previously24,25 demonstrated an excellent safety profile combined with induction of potent cellular immune responses assessed by ex vivo IFN-γ ELISPOT. Here, we present additional analyses of cellular immunogenicity across all three groups (i.e., Kenyan, Gambian, and UK adults) using both flow cytometry and IFN-γ ELISPOT on freshly isolated PBMC, as well as determination of humoral immunity by IgG enzyme-linked immunosorbent assay (ELISA) titer. Using this combination of simian adenovirus priming and poxvirus boosting vaccination, we demonstrate translation of the potent immunogenicity of vectored vaccines from malaria naive to semi-immune populations in Kenya and The Gambia.

Results

High-level immunogenicity following prime-boost vaccination in semi-immune adults

Prevaccination responses to both strains of the TRAP antigen were compared using the ex vivo IFN-γ ELISPOT assay, between the African trial sites and a previous UK trial with the same vectors to assess pre-existing immunity to the candidate vaccine antigen.24 No significant differences to either the T9/96 or 3D7 strain of P. falciparum (Figure 1a and Table 1) were observed between volunteers in the malaria-endemic regions where the field trials were performed and malaria-naive volunteers enrolled in a phase 2a controlled human malaria infection CHMI study of the same viral vectors in the United Kingdom.24

Figure 1.

Figure 1

Open in a new tab

Ex vivo IFN-γ ELISPOT responses pre- and postvaccination to ChAd63 and modified vaccinia Ankara (MVA) ME-TRAP-vectored vaccines in Gambian, Kenyan, and UK volunteers from a controlled human malaria infection trial vaccinated with the same construct. All volunteers received one dose of ChAd63 ME-TRAP followed by one dose of MVA ME-TRAP 8 weeks later. (a) Prevaccination responses to TRAP. (b) Responses to TRAP at 14 days postprime with ChAd63, ****P ≤ 0.0001, P = 0.03. (c) Responses to TRAP at day 63 (7 days postboost with MVA), ***P = 0.0002, **P = 0.003, *P = 0.04. (d) Responses to TRAP at day 300 postprime with ChAd63, ****P ≤ 0.0001, *P = 0.03. (e–g) Responses to the ME string in Gambian, Kenyan, and UK volunteers, *P < 0.05, **P < 0.01, ***P < 0.001. ELISPOT responses to peptides from different strains were compared a using two-tailed Wilcoxon-matched pairs analysis. Increases in ELISPOT responses to ME were compared to responses at day 0 using Kruskall-Wallis with Dunn's multiple comparison posttest. Lines represent medians. ELISPOT, enzyme-linked immunospot; TRAP, thrombospondin-related adhesive protein.

Table 1. Geometric mean IFN-γ ELISPOT responses by trial site, time point, and strain of Plasmodium falciparum used for TRAP peptide pool.

graphic file with name mt2014109t1.jpg

Open in a new tab

Priming vaccination with ChAd63 ME-TRAP induced modest T-cell responses after 14 days to peptides of the T9/96 strain of the TRAP antigen and these were slightly higher in Gambian than Kenyan vaccinees (group geometric means, 95% confidence intervals (CI) and P values for increases in ELISPOT response are shown in Table 1). Responses to the heterologous 3D7 strain of TRAP were significantly lower than those to the T9/96 vaccine strain in both trials at this time point (Figure 1b). Responses did not differ significantly to those in malaria naive adults.

Boosting with MVA ME-TRAP significantly increased the magnitude of IFN-γ ELISPOT responses to T9/96 in both trials, peaking at 1015 SFC/106 PBMC, (CI: 765–1,347) in the Gambian trial (P < 0.0001 compared to D14 responses) and 467 SFC/106 PBMC, (CI: 247–885; P = 0.0064) in Kenya. Peak responses in Gambians were higher than those in Kenyans (P = 0.04). When responses in vaccinees receiving the highest dose of ChAd63 (5 × 1010 viral particles (vp)) were combined across both trials, immunity to the T9/96 strain peaked at 1254 SFC/106 PBMC (CI: 912–1,724). Immunity to the 3D7 strain of TRAP also significantly increased after MVA vaccination to 901 SFC/106 PBMC in the Gambian (CI: 665–1,123; P < 0.0001) and 422 SFC/106 PBMC in Kenyan recipients (CI: 214–833; P = 0.0005; Figure 1c). For both strains, peak responses were lower in semi-immune adults than naive adults at the same time point, notably in Kenyans where the response was significantly lower (P < 0.01, Kruskall-Wallis test with Dunn's test for multiple comparisons, Table 1)

Immunogenicity was reassessed 8 months after MVA vaccination and plateaued at a level significantly higher than measured at baseline in Gambian participants. Responses in Kenyan volunteers at the same time point were only slightly higher than those detected prior to ChAd63 vaccination (Figure 1d).

Responses to the ME string of the ME-TRAP vaccine construct were substantially lower than to the TRAP antigen component. In The Gambia, responses peaked at 94 SFC/106 PBMC (CI: 66–132), Figure 1e and 105 SFC/106 PBMC (CI: 51–215) in Kenya, Figure 1f. These responses did not differ significantly from those observed in malaria-naive volunteers in the United Kingdom at the same time point (Figure 1g).

Vaccination induces broad responses to the TRAP antigen

At every time point studied, responses to the TRAP antigen were focused toward the first three peptide pools representing the N-terminal region, corresponding to amino acids (aa) 1-300, and the last peptide pool (aa 501-557) covering the C-terminal end. Responses to the central region (aa 300-500) of the antigen were rarely detected. The hierarchy of immunodominance for responses to the peptide pools was similar between malaria-naive and malaria-exposed vaccinees at each time point (Figure 2a–c). After each vaccination, the breadth of the immune response increased, as determined by the median number of peptide pools recognized, from 1 pool after ChAd63 vaccination in Kenyan participants to 3 after MVA vaccination. In Gambian participants, the breadth of response increased from 2 pools after ChAd63 to 3 at 1-week post-MVA and 4 pools at 5 weeks post-MVA.

Figure 2.

Figure 2

Open in a new tab

Percentage of vaccinees responding to each peptide pool. (a) prevaccination, (b) day 14 postprime with ChAd63 ME-TRAP, (c) 7 days postboost with MVA ME-TRAP. TRAP, thrombospondin-related adhesive protein.

Prime-boost immunization induces significant increases in IgG responses in both African trials

Humoral immunity to TRAP before and after vaccination was assessed by IgG ELISA using a protein of the 3D7 strain of TRAP. Pre-existing anti-TRAP IgG responses were compared between malaria-naive and exposed volunteers. Titers in the UK volunteers were significantly lower than those in both Gambian and Kenyan vaccinees (P < 0.001, Kruskall-Wallis test with Dunn's multiple comparison posttest, Figure 3a).

Figure 3.

Figure 3

Open in a new tab

Antibody responses and effect of pre-existing immunity on responses to vaccination. (a) Prevaccination anti-thrombospondin-related adhesive protein (TRAP) IgG responses, P < 0.001 for UK versus Kenya, P < 0.01 for UK versus The Gambia analyzed using Kruskal-Wallis with Dunn's multiple comparison test. Line represents geometric mean. (b) Geometric means of log endpoint titers for IgG ELISA against 3D7 TRAP stratified by dose of ChAd63. Dashed line represents 1 × 1010 viral particles (vp) and solid line 5 × 1010 vp ChAd63 ME-TRAP, P < 0.001, Kruskall-Wallis test with Dunn's multiple comparison posttest comparing responses at D14, D63, and D90 to baseline responses at D0. (c) Neutralizing antibody titers to the ChAd63 vector pre- and postimmunization at 14 days for African trials, ****P < 0.0001, paired two-tailed t-test. (d) Correlation between pre- and postimmunization neutralizing antibody titers. (e) Lack of association between cellular immune response to TRAP and antibody response to the ChAd63 vector at 14 days postimmunization. (f) Postprime and postboost immunization neutralizing antibody titers. (g) Correlation between pre-existing immunity to the ME-TRAP vaccine and responses at 14 days postvaccination with ChAd63. (h) Relationship between magnitude of response to priming vaccination at day 14 post-ChAd63 and boosting vaccination at day 63 (7 days post-MVA). Spearman's two-tailed correlation coefficients shown with P value. Upwards pointing triangles represent vaccinees primed with 1 × 1010 vp ChAd63 ME-TRAP in Kenya, downward pointing triangles 5 × 1010 vp in Kenya, circles 1 × 1010 vp ChAd63 ME-TRAP in The Gambia, and squares 5 × 1010 vp ChAd63 ME-TRAP in The Gambia.

There was no significant difference in antibody titer to TRAP between groups receiving low- or high-dose ChAd63 ME-TRAP at any time point in either trial (P > 0.05, two-tailed Mann–Whitney test, data not shown), and so, statistical analysis of increases was performed on combined data. Kinetics of antibody induction are shown in Figure 3b. Increases in antibody titers across both groups were significant after priming and boosting, (P < 0.001, Kruskall-Wallis test with Dunn's multiple comparison posttest comparing responses at D14 (Gambians only), D63, and D90 to baseline responses at D0). Peak responses at day 63 were 238 (CI: 159–358) ELISA units (EU) and 295 EU (CI: 235–370) in Gambian and Kenyan recipients respectively, and there was no significant difference in antibody response between sites (P = 0.3, two-tailed Mann–Whitney test).

Antibodies to the adenovirus vector do not inhibit responses to the vaccine antigen

Pre-existing immunity to ChAd63 was generally low in both trials, with geometric mean titer of 192 (CI: 147–251) in Gambians and 214 (CI: 104–442) in Kenyans (Figure 3c). Fourteen days after immunization with ChAd63 ME-TRAP, titers increased significantly (The Gambia 1,157, CI: 710–1,890; Kenya 1,255, CI: 696–2,264; P < 0.0001 using two-tailed t-test on log transformed data comparing pre- and postvaccination titers for each trial), and there was a strong positive correlation between anti-ChAd63 neutralizing antibody titers pre- and postvaccination (Spearman's r = 0.63, P < 0.0001, Figure 3d). There was a weak negative correlation between pre-existing humoral immunity to ChAd63 and T-cell responses to the vaccine at day 14 (Spearman's r = −0.3, P = 0.03, Figure 3e), however this association disappeared after boosting with MVA (Spearman's r = −0.2, P = 0.3, Figure 3f), suggesting that boosting of pre-existing immunity to the vector by vaccination was not associated with inhibition of cellular immunity to the vaccine. There was a trend toward a weak positive relationship between pre-existing cellular immunity to ME-TRAP and the response to ChAd63-ME-TRAP (Spearman's r = 0.3, P = 0.07, Figure 3g). After boosting with MVA, a highly significant relationship between T-cell responses measured postprime and postboost was apparent (Spearman's r = 0.6, P < 0.000 1, Figure 3h).

Dose of ChAd63 ME-TRAP and route of administration of MVA ME-TRAP do not affect immunogenicity

Cytokine secretion by CD4+ and CD8+ T cells was assessed by flow cytometry with intracellular cytokine staining using a single pool of peptides. Prior to vaccination, frequencies of cytokine-secreting T cells were low, with approximately equal distribution of cytokine production between CD4+ and CD8+ T cells in both trials (Supplementary Figure S2a). In Kenyan vaccinees, cytokine production was compared between volunteers vaccinated with MVA ME-TRAP via the intradermal (i.d.) and intramuscular (i.m.) routes. Prior to boosting with MVA, there were no significant differences in cytokine production between groups after correction for multiple comparisons for vaccinees primed with either low (1 × 1010 vp, Supplementary Figure S2b) or high (5 × 1010 vp, Supplementary Figure S2c) doses of ChAd63 ME-TRAP. After boosting, at day 63 (7 days post-MVA), no significant differences were detected for different routes of MVA administration in either of the dose groups (Supplementary Figure S2d,e), confirming the findings previously reported of the same responses assessed by IFN-γ ELISPOT. Therefore, subsequent analyses are presented with data for different routes of MVA administration combined.

In both African trials, the effect of priming dose of ChAd63 ME-TRAP on the magnitude of cytokine-secreting T-cell frequencies was assessed by flow cytometry. Irrespective of the dose of the priming vector used, IFN-γ was predominantly secreted by CD8+ T cells (pie-charts, Figure 4a,b). Secretion of IFN-γ, IL-2, and TNF-α could be detected from both CD4+ and CD8+ T cells at day 14, although responses tended to be higher in Kenyans. Seven days after boosting with MVA, IFN-γ secretion was more equally distributed between CD4+ and CD8+ T cells, particularly among vaccinees that received the higher dose of ChAd63 (pie-charts, Figure 4c,d).

Figure 4.

Figure 4

Open in a new tab

Responses to prime-boost immunization with ChAd63 and MVA ME-TRAP by ICS. Pie charts show proportion of IFN-γ secreted from CD4+ (light grey) and CD8+ (dark grey) as a proportion of total IFN-γ production. Scatter plots show individual responses to thrombospondin-related adhesive protein (TRAP) peptides from peripheral blood mononuclear cells stratified by trial and dose of priming immunization. Bars denote geometric mean. Cytokine frequencies from CD4+ (a) and CD8+ (b) T cells 14 days after priming immunization with ChAd63 ME-TRAP. c and d display cytokine secretion from CD4+ (c) and CD8+ (d) T cells after boosting with MVA ME-TRAP. Frequencies were compared with the Kruskall-Wallis test with Dunn's multiple comparison posttest, *P < 0.05, **P < 0.01. Upwards pointing triangles represent vaccinees primed with 1 × 1010 viral particles (vp) ChAd63 ME-TRAP in Kenya, downward pointing triangles 5 × 1010 vp in Kenya, circles 1 × 1010 vp ChAd63 ME-TRAP in The Gambia, and squares 5 × 1010 vp ChAd63 ME-TRAP in The Gambia. ICS, flow cytometry with intracellular cytokine staining; LLD, lower limit of detection of the assay.

No significant differences in magnitude of cytokine secreting T-cell populations were observed for different doses of ChAd63 within either trial; therefore, statistical analyses use data combined for both priming doses. At each trial site, frequencies of cytokine-secreting cells increased after each vaccination (Table 2 and scatter plots, Figure 4c,d). Within individual trial sites, increases were not statistically significant, however when data across trials were combined, increases in the magnitude of CD4+ IFN-γ+ and CD4+ TNFα+ populations reached significance (P = 0.007 and P = 0.009, Kruskall-Wallis test with Dunn's multiple comparison) and the increase in CD4+ IL2-secreting cells showed a trend toward an increase after vaccination (P = 0.07). Increases in CD8+ cytokine-secreting T-cell populations were not significant as frequencies of these populations were low. Analysis of polyfunctionality showed that a mixture of cell phenotypes was induced in all volunteers with single-, double-, and triple-cytokine secreting cells detected in both the CD4+ and CD8+ population (Figure 5a,b). CD8+ T cells secreting IFN-γ, but not IL-2 or TNF-α (mono-functional), which have been previously shown to associate with protection against CHMI with this vaccine regimen were detected from vaccinees in both trials, however the frequency in volunteers from the trial in Kenya was significantly higher than that in The Gambia or the United Kingdom (P = 0.008, Kruskall-Wallis with Dunn's multiple comparison correction, Figure 6).

Table 2. Geometric mean frequencies of cytokine-secreting T cells by trial site and time point.

graphic file with name mt2014109t2.jpg

Open in a new tab

Figure 5.

Figure 5

Open in a new tab

Polyfunctionality of peak T-cell responses at day 63. Analysis of cytokine secretion from CD4+ and CD8+ T cells was performed using a Boolean gating strategy in SPICE. Pie charts represent the proportion of the immune response represented by each combination of cytokines shown in the bar chart. Bar charts show the frequency of each population with bars representing median responses. (a) CD4+ responses. (b) CD8+ responses. SPICE, simplified presentation of incredibly complex evaluations.

Figure 6.

Figure 6

Open in a new tab

Frequency of monofunctional CD8+T cells (CD8+ T cells secreting IFN-γ, but not IL-2 or TNF-α) was significantly higher in Kenyan than Gambian or UK vaccinees. Frequencies were compared with the Kruskall-Wallis test with Dunn's multiple comparison posttest, *P < 0.05. Line shows geometric mean.

Discussion

Prime-boost vaccination with ChAd63 and MVA ME-TRAP has demonstrated excellent immunogenicity in these two phase 1b trials in healthy adults in regions of low-to-moderate malaria transmission. Despite presumably repeated exposure to malaria, pre-existing baseline T-cell responses to the TRAP antigen measured by ex vivo ELISPOT did not differ between malaria-naive and exposed volunteers, suggesting that TRAP is not a major target for naturally acquired cellular immunity. Similarly, after priming and boosting immunizations, T-cell responses to the vaccine antigen were of the same magnitude as those in UK malaria-naive vaccinees, suggesting that vaccination does not boost T-cell responses primed by natural exposure, despite the evidence of central memory responses to TRAP in Kenyan adults.19

The T9/96 and 3D7 strains of P. falciparum represent the breadth of genetic diversity of the TRAP antigen with 6.1% amino acid variation.26 Despite this, responses to the heterologous 3D7 strain (used for CHMI studies) were of a similar order of magnitude in both cohorts suggesting that this vaccine will not be affected substantially by antigenic polymorphism, as T cells induced by vaccination with the T9/96 strain were highly cross-reactive. Responses were detected to pools representing 46 of the 56 peptides spanning the TRAP protein (82%), demonstrating the high number of potential epitopes within this antigen. The breadth of the immune response induced by vaccination was also large and indicated that a number of epitopes over the length of the antigen were recognized. Given the potential diversity of HLA types in populations that might be targeted with a malaria vaccine, the finding of potentially highly promiscuous epitopes is encouraging.

More than a decade ago, the approach of combining a DNA priming vaccination with MVA boosting using the same ME-TRAP construct induced very modest T-cell responses by ex vivo ELISPOT (GM 69.8 SFC/106 PBMC) that failed to reduce the rate of malaria infection in Gambian men,27,28 most likely as a result of insufficient immunogenicity. Previous trials of prime-boost viral vectored vaccines with this antigen that employed two pox viruses (albeit different viruses) as both priming and boosting vectors failed to elicit sufficiently potent immunity to mediate protection in volunteers in coastal Kenya or The Gambia.18,19,20 Although the immunogenicity induced by the fowlpox-MVA ME-TRAP vaccination regime was more immunogenic than DNA-MVA, the peak response was still modest at 185 SFC/106 PBMC. After a double prime with fowlpox ME-TRAP, peak responses reached 389 SFC/106 PBMC, however this was also insufficient to induce protection against febrile malaria among young children in Kenya. In adults, immunogenicity in both settings was lower than in malaria-naive adults, most likely due to immunosuppression caused by malaria. Data from Kenyan children showed reduced T-cell responses after both vaccination with poxviruses, and natural exposure was associated with parasitaemia and clinical malaria.29

Here, we show that replacement of the fowlpox or DNA priming vectors with the chimpanzee adenovirus vector has both increased the magnitude of the peak response fourfold to 1254 SFC per million PBMC and resulted in increased induction of CD8+ T-cell-mediated immunity—a prerequisite for an effective pre-erythrocytic vaccine.30 This suggests that adenoviral vectors may be more robust to the immunosuppressive effects of malaria than poxvirus vectors, a potentially useful characteristic for vaccines against other diseases in malaria-endemic regions. In addition, responses were maintained above baseline in the Kenyan vaccinees for 8 months, demonstrating useful durability of effector memory type cellular immunity and potentially greater longitudinal central memory. Vaccination with ChAd63 and MVA ME-TRAP also induced modest, but significant increases in TRAP IgG levels, despite a higher level of pre-existing humoral immunity than in malaria-naive vaccinees, suggesting that prior immunity to TRAP does not inhibit development of vaccine-induced antibodies.

Contrary to prevailing theory, pre-existing immunity to this adenoviral vector did not inhibit the subsequent immunogenicity of the vaccine, in fact a trend toward a weak positive association was detected. Our group has previously demonstrated a much stronger positive association in UK volunteers, however the finding reported here in regions of increased prevalence of neutralizing antibodies to the adenoviral vector is pertinent for the potential deployment of this vaccine in an endemic population and similar levels of seroprevalence to this vector have recently been reported in Burkina Faso.31 Given the strong correlation between responses after priming and boosting, the magnitude of the peak response is clearly related to the magnitude of the response to ChAd63 ME-TRAP, rather than any humoral response to the adenoviral vector.

The dose of ChAd63 used for priming did not significantly affect the immunogenicity of the vaccine by any of the measures we studied, in agreement with the first UK phase 1 dose-escalation study of the ChAd63 ME-TRAP priming immunization.21 Furthermore, administration of the boosting MVA ME-TRAP vector by either the i.m. or i.d. routes had no effect on responses to the TRAP insert. Given that i.m. immunization is easier to perform with reduced local reactogenicity,25 future trials of the MVA ME-TRAP vaccine will employ this route of administration.

Phenotyping of vaccine-induced T-cell subtypes by flow cytometry with intracellular cytokine staining revealed production of IFN-γ, IL-2, and TNF-α from CD4+ and CD8+ T cells in all combinations. In a previously reported study of the efficacy of ChAd63 and MVA ME-TRAP in the United Kingdom, we showed 21% sterile efficacy increasing to more than 50% efficacy when including delay to detectable parasitaemia.24 We then demonstrated a correlation between efficacy and frequency of monofunctional CD8+ T cells secreting IFN-γ, a finding also observed in a nonhuman primate model of sporozoite-induced protection.32 In this study, the frequency of this protective CD8+ T-cell population was significantly higher in Kenyan vaccinees than in the UK study, suggesting that this vaccine may be more protective in this population, particularly given that the infectious inoculum during natural exposure may well be lower than in the CHMI model of sporozoite challenge.33

Although practical limitations prevented flow cytometric analysis of the memory phenotype of the vaccine-induced T-cell response, previous studies in mice using the same vaccine regimen clearly identified CD8+ T effector memory T cells as essential for protection against the pre-erythrocytic stage of malaria.12 In humans, the exact nature of T-cell phenotype best suited to mediating durable protective immunity is not clear, with different models proposing both effector and central memory T cell as more important.34,35 Future studies of vaccine efficacy could address these conflicting positions, although markers for discrimination of effector and memory populations in human T cells are complex due to the heterogeneous nature of the phenotypes.36

The initial report of these two phase 1 clinical trials demonstrated excellent safety with very acceptable reactogenicity profiles for both ChAd63 and MVA ME-TRAP.25 In this report, we have detailed the very potent cellular immunogenicity of this vaccine for two increasing doses of the ChAd63 priming vector and two routes of administration for the MVA vector. This regimen elicits a broad CD4+ and CD8+ T-cell-mediated immune response including a phenotype shown to induce protective efficacy against sporozoite challenge. The combination of an acceptable safety profile and impressive immunogenicity provides a clear rationale for phase 1 age de-escalation studies in children in semi-immune populations and phase 2b efficacy studies in adults in endemic regions. Studies of both such designs are underway. The data presented here also highlight the potential utility of these viral vectors as vaccines for other diseases where induction of high levels of T-cell immunity is helpful or necessary for protection.

Materials and Methods

The objectives, setting, participant demographics, clinical study design, and methods for vaccine production used in these clinical trials have been described in detail elsewhere.25 Brief details are repeated here.

Study settings and design. The first trial was conducted at the Medical Research Council field site located within Sukuta Health Centre in Kombo North district of The Gambia, West Africa. Sukuta village has an estimated population of 17,000 (2003 census). The climate is typical of sub-Saharan Africa with a long dry season lasting from December to June followed by a relatively short rainy season from July to November when the majority of P. falciparum malaria transmission occurs.37

The second trial was conducted in Vipingo, Kilifi County, Kenya, East Africa. Participants were recruited from the Rea Vipingo Sisal Plantation Estates in Kilifi, which has over 1,000 employees and a land area of 3,950 hectares. In Kilifi, there are two seasons of high transmission of P. falciparum malaria coinciding with the long monsoon rains (April to June) and the short rains (October to December).38

The third trial was conducted in Oxford, UK, and local malaria-naive volunteers were recruited.

We conducted two phase 1b open-label malaria vaccine trials. Both trials evaluated low- (1 × 1010 vp) and high-dose (5 × 1010 vp) ChAd63 ME-TRAP. The trial in Kenya also compared i.m. and i.d. routes of administration of 2 × 108 p.f.u. MVA ME-TRAP. In The Gambia, eligible participants were allocated to receive either ChAd63 ME-TRAP 1 × 1010 vp (group 1; n = 6) or ChAd63 ME-TRAP 5 × 1010 vp (group 2; n = 10) administered i.m. in the deltoid. All participants were subsequently vaccinated in the opposite arm 56 days later with 2 × 108 plaque forming units (p.f.u.) MVA ME-TRAP administered i.m.. In the Kenyan trial, eligible participants were allocated to receive either ChAd63 ME-TRAP 5 × 109 vp (group 1; n = 10) or ChAd63 ME-TRAP 5 × 1010 vp (group 2; n = 20) administered i.m. in the deltoid. All participants were subsequently vaccinated in the opposite arm 56 days later with 2 × 108 p.f.u. MVA ME-TRAP. Participants in each group were randomized 1:1 to receive MVA ME-TRAP administered i.m. or i.d. Blood samples for immunology were collected on day 0 (day of ChAd63 ME-TRAP vaccination), day 14, day 28, day 56 (day of MVA ME-TRAP vaccination), day 63, day 90, and day 300 (The Gambia) or day 308 (Kenya).

The UK trial was a phase 2a sporozoite challenge study (CHMI) with volunteers receiving ChAd63 ME-TRAP 5 × 1010 vp administered i.m. followed by of 2 × 108 p.f.u. MVA ME-TRAP administered i.d. 56 days later.24 Data shown here are from blood samples collected for immunology on day 0, 14, 28, 56, and 63; no data are shown post-CHMI. Some data were reanalyzed for comparability with methods employed in this study. In particular, the IgG antibody data shown here were performed using a standardized anti-TRAP ELISA rather than the end-point titer assay described in the previous publication, however agreement between the two methods was excellent and data correlated extremely well (Spearman's r = 0.9, two-tailed P < 0.0001).

Ethical and regulatory approval. The clinical trial protocols and associated documents were approved by Gambia Government/Medical Research Council Joint Ethics Committee and The Kenya Medical Research Institute National Ethics Committee, respectively. Documents for all clinical trials were reviewed and approved by the Oxford Tropical Research Ethics Committee. Regulatory approval was given by the Medicines Board of The Gambia, The Pharmacy and Poisons Board of Kenya, and the Medicines and Healthcare products Regulatory Agency, respectively. All participants gave documented informed consent prior to any study procedure being undertaken. The study was conducted according to the principles of the Declaration of Helsinki (2008) and the International Conference on Harmonization Good Clinical Practice guidelines. An independent Data and Safety Monitoring Board and local safety monitors provided safety oversight and Good Clinical Practice compliance was independently monitored by an external organization at both trial sites (Appledown Clinical Research, Great Missenden, UK). Trials were registered with Pan African Clinical Trial Registry (Ref: PACT ATMR 2010020001771828 & PACTR 201008000221638); http://www.pactr.org/ and ClinicalTrials.gov (Ref: NCT01373879, NCT01379430 & NCT00890760); http://clinicaltrials.gov/ct2/home.

Standardization of immunological methods. Staff from the Kenyan and Gambian immunology laboratories visited The Jenner Institute for training on immunology protocols that were standardized across all three sites. Staff from the Jenner Institute visited both African institutes to help with sample processing and equipment optimization. For cellular immunology, reagents were provided from The Jenner Institute after batch testing and the same batch of peptides were used in all studies. ELISAs and neutralizing antibody assays on samples from The Gambia and UK studies were performed at the Jenner Institute and at KEMRI for the Kenyan study using reagents supplied by the Jenner Institute. Data analysis was largely performed independently at each site with quality control performed by a single senior scientist at the Jenner Institute. All assays of cellular immunity reported here were performed on freshly isolated PBMC.

Ex vivo ELISPOT assays. Ex vivo (18-hour stimulation) ELISPOT assays were performed using Multiscreen IP ELISPOT plates (Merck Millipore, Billerica, MA), human IFN-γ SA-ALP antibody kits (Mabtech, Stockholm, Sweden), and BCIP NBT-plus chromogenic substrate (Moss, Pasadena, MD). Cells were cultured in RPMI (Sigma, Poole, UK) containing 10% heat-inactivated, sterile-filtered fetal calf serum, previously screened for low reactivity (Labtech International, Uckfield, UK). Antigens were tested in duplicate with 250,000 freshly isolated PBMC added to each well of the ELISPOT plate.16 TRAP peptides were 20 amino acids in length, overlapping by 10 amino acids (Neopeptide, Cambridge, MA), assayed in 6 pools of 7–10 peptides at 10 μg/ml. Responses were averaged across duplicates, responses in unstimulated (negative control) wells were subtracted, and then responses in individual pools were summed for each strain of the TRAP antigen. ME responses were assayed in a single pool and peptide pool configurations are shown in Supplementary Tables S1 and S2. Staphylococcal enzyme B (SEB, 0.02μg/ml) and phytohemmagglutinin-L (10 μg/ml, Sigma) were used as a positive control. Plates were counted using an AID automated ELISPOT counter (AID Diagnostika GmbH, Strassberg, Germany; algorithm C), using identical settings for all plates, and counts were adjusted only to remove artifacts. Responses to the negative control were always less than 90 SFC per million PBMC and the median across both trials was 11 SFC per million PBMC. Pools were considered positive if the response was greater than 11 SFC per million PBMC and four times higher than the negative control for that assay. The lower limit of detection for the assay is 24 SFC per million PBMC for peptide pools and 4 SFC for individual pools.

Flow cytometry. PBMC were separated by density centrifugation from heparinized whole blood and resuspended in polypropylene tubes at 1 × 106 PBMC per ml in RPMI containing 10% heat-inactivated, batch-tested, sterile-filtered fetal bovine serum, 1% L-glutamine, 1% penicillin/streptomycin, and 1 μg/ml each of anti-CD28 and anti-CD49d (BD Biosciences, Oxford, UK). Samples were stored at room temperature prior to processing, which was completed within 6 hours of venipuncture. Cell counts were performed using trypan blue staining and a microscope according to an established SOP in the laboratory. Cells were then stimulated overnight at 37 °C with 5% CO2, either with a pool of 56 peptides at 2 μg/ml spanning the entire length of the TRAP protein from the T9/96 strain of P. falciparum comprising 20mers overlapping by 10 amino acids, SEB or an unstimulated control containing an equivalent concentration of DMSO to the TRAP peptide pool (0.3%). Brefeldin A (BD Biosciences) was after 2 hours into the incubation at 1 μg/ml. Cells were then washed in fluorescence-activated cell sorting buffer (phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA) and 0.01% sodium azide, Sigma) and stained for viability with LIVE/DEAD aqua amine reactive dye (Life technologies, Carlsbad, CA) for 20 minutes at 4 °C in the dark, after which a cocktail of antibodies for surface marker staining was added and incubated for 30 minutes at 4 °C in the dark. Cells were washed again in fluorescence-activated cell sorting buffer, then permeabilized for 30 minutes with Cytofix/Cytoperm (BD Biosciences), and washed in permeabilization buffer (BD Biosciences). A cocktail of antibodies for intracellular staining was then added and incubated for 30 minutes at room temperature in the dark. Antibody cocktails are described in Supplementary Table S3. Cells were washed in permeabilization buffer and resuspended in PBS containing 1% paraformaldehyde, prior to acquisition on CyAn flow cytometers (Beckman Coulter, High Wycombe, UK) on the day of staining. Compensation control beads (Compbeads, BD Biosciences) were stained according to the manufacturers instructions using the same concentration of antibody used for cells to for compensation between parameters, and unstained cells were used to adjust forward and side scatter photo-multiplier tube voltages.

Analysis of flow cytometry data. At least 50,000 live CD3+ cells were analyzed per sample. Data were prepared and analyzed using FlowJo v8.8.6 (Treestar, Ashland, Oregon), Pestle v1.6 and Spice v5.05 (Mario Roederer, Vaccine Research Centre, NIAID, NIH). A hierarchical gating strategy was used as described in Supplementary Figure S1. Responses to peptide were determined after subtraction of the response in the unstimulated control for each sample. Pie charts were created using absolute measures with a threshold of 0.002%. For analyses of multiple cytokine function, all samples had >10,000 CD4+ or CD8+ T cells in the parent population. Samples where a response to the positive control of greater than 1% cytokine positive CD4+ or CD8+ T cells could not be detected were excluded from the analysis. The lower limit of detection for the assay was 0.002%, and a positive response was greater than two times the medium control for the corresponding sample.

Antivector neutralizing antibody assay. One day prior to performing the assay, GripTite 293 cells (Life Technologies) were seeded in 96-well plates (3 × 104 cells/well). Heat-inactivated test samples were diluted fourfold from 1:9 to 1:2,304 in 10% sterile-filtered fetal bovine serum in Dulbecco's modified Eagle's medium and incubated 1:1 with ChAd63 expressing the secreted alkaline phosphatase gene (8 × 107 vp/ml) for 1 hour at 37 °C. Serum and virus were then added to 293 cells in a volume of 200 µl in duplicate for 1 hour, after which sample and virus were aspirated and replaced with fresh 10% fetal bovine serum Dulbecco's modified Eagle's medium. A virus-only control was included. After 22–26 hours at 37 °C, 50 µl of medium was assayed for SEAP activity using a Phospha-Light TROPIX phosphatase assay (Life Technologies) in black assay plates, and luminescence was measured after 45 minutes on a Varioskan Flash Luminometer (ThermoFisher, Waltham, MA). Antivector neutralization titers were defined as the dilution of serum showing 50% reduction in SEAP activity, based on observed % inhibition values relative to SEAP activity from virus alone. Antivector antibodies were measured in serum in The Gambia and plasma for Kenyans, according to sample availability and local clinical practice, however we have determined that these sample types are equivalent for this assay.

Serum IgG antibody responses. Antibody responses were measured by anti-TRAP IgG sandwich ELISA. Nunc-Immuno 96 well plates were coated with 0.5 μg/ml of TRAP antigen in carbonate-bicarbonate coating buffer and left overnight at 4 °C. Plates were washed 6× with PBS-Tween (PBS/T), then blocked with 1% BSA in PBS/T for 1 hour at room temperature. Serum or plasma was diluted in PBS/T containing 0.2% BSA at concentrations of 1:100, 1:500, or 1:1,000, and added in triplicate. Samples from days 0 (ChAd63 vaccination), 28, 56 (MVA vaccination), 63, and 300 were analyzed. Plates were incubated at room temperature for 2 hours; then washed as before. A secondary antibody (goat anti-human whole IgG conjugated to alkaline phosphatase, Sigma) was added at a dilution of 1:1,000 in PBS/T 0.2% BSA for 1 hour at room temperature. After a final wash, plates were developed by adding 4-nitrophenyl phosphate in diethanolamine buffer (Pierce, Loughborough, UK).

A positive reference standard (made from pooled TRAP-positive serum) was used on each plate to give a standard curve. It was added in duplicate at an initial dilution of 1:100 (in PBS/T 0.2% BSA) and diluted twofold 10 times, starting with an arbitrary value of 20 antibody units. Four blank wells (zero antibody units) were also designated. The optical density values were then fitted to a 4 parameter standard curve using SOFTmax PRO software.15 An internal control was included on every plate in triplicate made up from a 1:800 dilution (in PBS/T 0.2% BSA) of the positive standard. Optical density was read at 405 nm using an ELx800 microplate reader (Cole Parmer, London, UK). Test sera antibody units were calculated from their optical density values using the parameters estimated from the standard curve.

Statistical analysis. Group data are geometric means unless otherwise stated with 95% CI. The matched pairs analysis excludes volunteers with missing data at any time point. A Kruskall-Wallis test was used to compare increases in T-cell frequencies in time courses with Dunn's multiple comparisons posttest used to compare response pre- and postvaccination. For statistical analyses, an α-level of 0.05 was considered significant and all P values are two-tailed. All analyses were performed in GraphPad Prism, Mac version 5 (GraphPad, La Jolla, CA).

SUPPLEMENTARY MATERIAL Figure S1. Sample gating strategy for flow cytometry analysis. Figure S2. Effect of route of MVA administration on responses to TRAP peptides by ICS, expressed as the percentage of cytokine-secreting CD4+ or CD8+ T cells. Table S1. TRAP peptide pool format. Table S2. ME peptide pool format. Table S3. Reagents used for flow cytometry staining.

Acknowledgments

For the study in The Gambia, we thank the Gambian National Malaria Control Programme and staff of Sukuta Health Centre for their collaboration; J Adetifa and the Sukuta field team; E Touray, S Sanneh, M Sambou, and O Badjie for field work; J Jobe, F Darboe, and Isatou Bah for laboratory assistance; O Nyan for safety monitoring; J Suso for clinical trial support; M Sowe and H Kanyi for help with data management and C McKenna for study monitoring. For the study in Kenya, we thank V Mwatemo for clinical assistance, REA Vipingo Plantations Ltd for use of facilities, C McKenna for study monitoring, M Otieno for data management, A Etyang' for safety monitoring, and E Pembe and A Mbaru for field work. We thank the members of both Data Safety Monitoring Boards and all the study volunteers. We thank W Ripley Ballou of GSK for reviewing the manuscript. This study is published with the permission of the Director of the Kenyan Medical Research Institute. This work was supported by an award from the European and Developing Countries Clinical Trials Partnership (EDCTP) and was performed by the Malaria Vectored Vaccines Consortium (MVVC), a four and a half year integrated project funded by the European and Developing Countries Clinical Trials Partnership (EDCTP, grant number IP.2008.31100.001). N.I.V. is an employee of the European Vaccine Initiative (EVI). E.V.I. is the coordinator of the EDCTP funded MVVC project (grant number IP.2008.31100.001). E.V.I. supports salaries of the MVVC project in kind. The work was also supported by the UK National Institute of Health Research through the Oxford Biomedical Research Centre (http://www.oxfordbrc.org/) [A91301 Adult Vaccine], the Wellcome Trust (http://www.wellcome.ac.uk/) (084113/Z/07/Z) and the Medical Research Council. S.H.H. holds a Wellcome Trust research training fellowship (097940/Z/11/Z). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. A.V.S.H. is a named inventor on patent applications on malaria vectored vaccines and immunization regimens. Authors from Okairòs are employees of and/or shareholders in Okairòs, which is developing vectored vaccines for malaria and other diseases.

Supplementary Material

Supplementary Figure S1

Sample gating strategy for flow cytometry analysis.

Click here for additional data file. (745.8KB, tiff)
Supplementary Figure S2

Effect of route of MVA administration on responses to TRAP peptides by ICS, expressed as the percentage of cytokine-secreting CD4+ or CD8+ T cells.

Click here for additional data file. (354.5KB, tiff)
Supplementary Table S1

TRAP peptide pool format.

Click here for additional data file. (104.5KB, doc)
Supplementary Table S2

ME peptide pool format.

Click here for additional data file. (39KB, doc)
Supplementary Table S3

Reagents used for flow cytometry staining.

Click here for additional data file. (36KB, doc)

References

  1. WHO . World Malaria Report 2013. WHO; Geneva; 2013. [Google Scholar]
  2. Noor AM, Kinyoki DK, Mundia CW, Kabaria CW, Mutua JW, Alegana VA, et al. The changing risk of Plasmodium falciparum malaria infection in Africa: 2000-10: a spatial and temporal analysis of transmission intensity. Lancet. 2014;383:1739–1747. doi: 10.1016/S0140-6736(13)62566-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. White NJ, Pukrittayakamee S, Hien TT, Faiz MA, Mokuolu OA, Dondorp AM. Malaria. Lancet. 2014;383:723–735. doi: 10.1016/S0140-6736(13)60024-0. [DOI] [PubMed] [Google Scholar]
  4. Malaria Vaccine Funders Group 2013Malaria Vaccine Technology Roadmap . http://www.who.int/immunization/topics/malaria/vaccine_roadmap . Accessed 30 June 2014.
  5. Kester KE, Cummings JF, Ofori-Anyinam O, Ockenhouse CF, Krzych U, Moris P, et al. RTS,S Vaccine Evaluation Group Randomized, double-blind, phase 2a trial of falciparum malaria vaccines RTS,S/AS01B and RTS,S/AS02A in malaria-naive adults: safety, efficacy, and immunologic associates of protection. J Infect Dis. 2009;200:337–346. doi: 10.1086/600120. [DOI] [PubMed] [Google Scholar]
  6. RTSS Clinical trials partnership A phase 3 trial of RTS,S/AS01 malaria vaccine in African infants. N Engl J Med. 2012;367:2284–2295. doi: 10.1056/NEJMoa1208394. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. White MT, Bejon P, Olotu A, Griffin JT, Riley EM, Kester KE, et al. The relationship between RTS,S vaccine-induced antibodies, CD4(+) T cell responses and protection against Plasmodium falciparum infection. PLoS One. 2013;8:e61395. doi: 10.1371/journal.pone.0061395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ndungu FM, Mwacharo J, Kimani D, Kai O, Moris P, Jongert E, et al. A statistical interaction between circumsporozoite protein-specific T cell and antibody responses and risk of clinical malaria episodes following vaccination with RTS,S/AS01E. PLoS ONE. 2012;7:e52870. doi: 10.1371/journal.pone.0052870. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Schmidt NW, Butler NS, Badovinac VP, Harty JT. Extreme CD8 T cell requirements for anti-malarial liver-stage immunity following immunization with radiation attenuated sporozoites. PLoS Pathog. 2010;6:e1000998. doi: 10.1371/journal.ppat.1000998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Weiss WR, Jiang CG. Protective CD8+ T lymphocytes in primates immunized with malaria sporozoites. PLoS ONE. 2012;7:e31247. doi: 10.1371/journal.pone.0031247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Schmidt NW, Butler NS, Harty JT. CD8 T cell immunity to Plasmodium permits generation of protective antibodies after repeated sporozoite challenge. Vaccine. 2009;27:6103–6106. doi: 10.1016/j.vaccine.2009.08.025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Reyes-Sandoval A, Wyllie DH, Bauza K, Milicic A, Forbes EK, Rollier CS, et al. CD8+ T effector memory cells protect against liver-stage malaria. J Immunol. 2011;187:1347–1357. doi: 10.4049/jimmunol.1100302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Bijker EM, Bastiaens GJ, Teirlinck AC, van Gemert GJ, Graumans W, van de Vegte-Bolmer M, et al. Protection against malaria after immunization by chloroquine prophylaxis and sporozoites is mediated by preerythrocytic immunity. Proc Natl Acad Sci USA. 2013;110:7862–7867. doi: 10.1073/pnas.1220360110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Birkett AJ, Moorthy VS, Loucq C, Chitnis CE, Kaslow DC. Malaria vaccine R&D in the Decade of Vaccines: breakthroughs, challenges and opportunities. Vaccine. 2013;31 suppl. 2:B233–B243. doi: 10.1016/j.vaccine.2013.02.040. [DOI] [PubMed] [Google Scholar]
  15. McConkey SJ, Reece WH, Moorthy VS, Webster D, Dunachie S, Butcher G, et al. Enhanced T-cell immunogenicity of plasmid DNA vaccines boosted by recombinant modified vaccinia virus Ankara in humans. Nat Med. 2003;9:729–735. doi: 10.1038/nm881. [DOI] [PubMed] [Google Scholar]
  16. Webster DP, Dunachie S, Vuola JM, Berthoud T, Keating S, Laidlaw SM, et al. Enhanced T cell-mediated protection against malaria in human challenges by using the recombinant poxviruses FP9 and modified vaccinia virus Ankara. Proc Natl Acad Sci USA. 2005;102:4836–4841. doi: 10.1073/pnas.0406381102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Imoukhuede EB, Berthoud T, Milligan P, Bojang K, Ismaili J, Keating S, et al. Safety and immunogenicity of the malaria candidate vaccines FP9 CS and MVA CS in adult Gambian men. Vaccine. 2006;24:6526–6533. doi: 10.1016/j.vaccine.2006.06.022. [DOI] [PubMed] [Google Scholar]
  18. Imoukhuede EB, Andrews L, Milligan P, Berthoud T, Bojang K, Nwakanma D, et al. Low-level malaria infections detected by a sensitive polymerase chain reaction assay and use of this technique in the evaluation of malaria vaccines in an endemic area. Am J Trop Med Hyg. 2007;76:486–493. [PMC free article] [PubMed] [Google Scholar]
  19. Bejon P, Kai OK, Mwacharo J, Keating S, Lang T, Gilbert SC, et al. Alternating vector immunizations encoding pre-erythrocytic malaria antigens enhance memory responses in a malaria endemic area. Eur J Immunol. 2006;36:2264–2272. doi: 10.1002/eji.200636187. [DOI] [PubMed] [Google Scholar]
  20. Bejon P, Mwacharo J, Kai O, Mwangi T, Milligan P, Todryk S, et al. A phase 2b randomised trial of the candidate malaria vaccines FP9 ME-TRAP and MVA ME-TRAP among children in Kenya. PLoS Clin Trials. 2006;1:e29. doi: 10.1371/journal.pctr.0010029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. O'Hara GA, Duncan CJ, Ewer KJ, Collins KA, Elias SC, Halstead FD, et al. Clinical assessment of a recombinant simian adenovirus ChAd63: a potent new vaccine vector. J Infect Dis. 2012;205:772–781. doi: 10.1093/infdis/jir850. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Sheehy SH, Duncan CJ, Elias SC, Collins KA, Ewer KJ, Spencer AJ, et al. Phase Ia clinical evaluation of the Plasmodium falciparum blood-stage antigen MSP1 in ChAd63 and MVA vaccine vectors. Mol Ther. 2011;19:2269–2276. doi: 10.1038/mt.2011.176. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. de Cassan SC, Forbes EK, Douglas AD, Milicic A, Singh B, Gupta P, et al. The requirement for potent adjuvants to enhance the immunogenicity and protective efficacy of protein vaccines can be overcome by prior immunization with a recombinant adenovirus. J Immunol. 2011;187:2602–2616. doi: 10.4049/jimmunol.1101004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Ewer KJ, O'Hara GA, Duncan CJ, Collins KA, Sheehy SH, Reyes-Sandoval A, et al. Protective CD8+ T-cell immunity to human malaria induced by chimpanzee adenovirus-MVA immunisation. Nat Commun. 2013;4:2836. doi: 10.1038/ncomms3836. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ogwang C, Afolabi M, Kimani D, Jagne YJ, Sheehy SH, Bliss CM, et al. Safety and immunogenicity of heterologous prime-boost immunisation with Plasmodium falciparum malaria candidate vaccines, ChAd63 ME-TRAP and MVA ME-TRAP, in healthy Gambian and Kenyan adults. PLoS ONE. 2013;8:e57726. doi: 10.1371/journal.pone.0057726. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Robson KJ, Dolo A, Hackford IR, Doumbo O, Richards MB, Keita MM, et al. Natural polymorphism in the thrombospondin-related adhesive protein of Plasmodium falciparum. Am J Trop Med Hyg. 1998;58:81–89. doi: 10.4269/ajtmh.1998.58.81. [DOI] [PubMed] [Google Scholar]
  27. Moorthy VS, Imoukhuede EB, Milligan P, Bojang K, Keating S, Kaye P, et al. A randomised, double-blind, controlled vaccine efficacy trial of DNA/MVA ME-TRAP against malaria infection in Gambian adults. PLoS Med. 2004;1:e33. doi: 10.1371/journal.pmed.0010033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Moorthy VS, Pinder M, Reece WH, Watkins K, Atabani S, Hannan C, et al. Safety and immunogenicity of DNA/modified vaccinia virus ankara malaria vaccination in African adults. J Infect Dis. 2003;188:1239–1244. doi: 10.1086/378515. [DOI] [PubMed] [Google Scholar]
  29. Bejon P, Mwacharo J, Kai O, Todryk S, Keating S, Lowe B, et al. The induction and persistence of T cell IFN-gamma responses after vaccination or natural exposure is suppressed by Plasmodium falciparum. J Immunol. 2007;179:4193–4201. doi: 10.4049/jimmunol.179.6.4193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Robinson HL, Amara RR. T cell vaccines for microbial infections. Nat Med. 2005;11 suppl. 4:S25–S32. doi: 10.1038/nm1212. [DOI] [PubMed] [Google Scholar]
  31. Nébié I, Edwards NJ, Tiono AB, Ewer KJ, Sanou GS, Soulama I, et al. Assessment of chimpanzee adenovirus serotype 63 neutralizing antibodies prior to evaluation of a candidate malaria vaccine regimen based on viral vectors. Clin Vaccine Immunol. 2014;21:901–903. doi: 10.1128/CVI.00723-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Epstein JE, Tewari K, Lyke KE, Sim BK, Billingsley PF, Laurens MB, et al. Live attenuated malaria vaccine designed to protect through hepatic CD8(+) T cell immunity. Science. 2011;334:475–480. doi: 10.1126/science.1211548. [DOI] [PubMed] [Google Scholar]
  33. Sauerwein RW, Roestenberg M, Moorthy VS. Experimental human challenge infections can accelerate clinical malaria vaccine development. Nat Rev Immunol. 2011;11:57–64. doi: 10.1038/nri2902. [DOI] [PubMed] [Google Scholar]
  34. Wherry EJ, Teichgräber V, Becker TC, Masopust D, Kaech SM, Antia R, et al. Lineage relationship and protective immunity of memory CD8 T cell subsets. Nat Immunol. 2003;4:225–234. doi: 10.1038/ni889. [DOI] [PubMed] [Google Scholar]
  35. Olson JA, McDonald-Hyman C, Jameson SC, Hamilton SE. Effector-like CD8(+) T cells in the memory population mediate potent protective immunity. Immunity. 2013;38:1250–1260. doi: 10.1016/j.immuni.2013.05.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Jameson SC, Masopust D. Diversity in T cell memory: an embarrassment of riches. Immunity. 2009;31:859–871. doi: 10.1016/j.immuni.2009.11.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Greenwood BM, Pickering H. A malaria control trial using insecticide-treated bed nets and targeted chemoprophylaxis in a rural area of The Gambia, west Africa. 1. A review of the epidemiology and control of malaria in The Gambia, west Africa. Trans R Soc Trop Med Hyg. 1993;87 suppl. 2:3–11. doi: 10.1016/0035-9203(93)90169-q. [DOI] [PubMed] [Google Scholar]
  38. Mwangi TW, Ross A, Snow RW, Marsh K. Case definitions of clinical malaria under different transmission conditions in Kilifi District, Kenya. J Infect Dis. 2005;191:1932–1939. doi: 10.1086/430006. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Figure S1

Sample gating strategy for flow cytometry analysis.

Click here for additional data file. (745.8KB, tiff)
Supplementary Figure S2

Effect of route of MVA administration on responses to TRAP peptides by ICS, expressed as the percentage of cytokine-secreting CD4+ or CD8+ T cells.

Click here for additional data file. (354.5KB, tiff)
Supplementary Table S1

TRAP peptide pool format.

Click here for additional data file. (104.5KB, doc)
Supplementary Table S2

ME peptide pool format.

Click here for additional data file. (39KB, doc)
Supplementary Table S3

Reagents used for flow cytometry staining.

Click here for additional data file. (36KB, doc)

Articles from Molecular Therapy are provided here courtesy of The American Society of Gene & Cell Therapy

RESOURCES