• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of moltherLink to Publisher's site
Mol Ther. Dec 2012; 20(12): 2355–2368.
Published online Oct 23, 2012. doi:  10.1038/mt.2012.223
PMCID: PMC3519995

ChAd63-MVA–vectored Blood-stage Malaria Vaccines Targeting MSP1 and AMA1: Assessment of Efficacy Against Mosquito Bite Challenge in Humans

Abstract

The induction of cellular immunity, in conjunction with antibodies, may be essential for vaccines to protect against blood-stage infection with the human malaria parasite Plasmodium falciparum. We have shown that prime-boost delivery of P. falciparum blood-stage antigens by chimpanzee adenovirus 63 (ChAd63) followed by the attenuated orthopoxvirus MVA is safe and immunogenic in healthy adults. Here, we report on vaccine efficacy against controlled human malaria infection delivered by mosquito bites. The blood-stage malaria vaccines were administered alone, or together (MSP1+AMA1), or with a pre-erythrocytic malaria vaccine candidate (MSP1+ME-TRAP). In this first human use of coadministered ChAd63-MVA regimes, we demonstrate immune interference whereby responses against merozoite surface protein 1 (MSP1) are dominant over apical membrane antigen 1 (AMA1) and ME-TRAP. We also show that induction of strong cellular immunity against MSP1 and AMA1 is safe, but does not impact on parasite growth rates in the blood. In a subset of vaccinated volunteers, a delay in time to diagnosis was observed and sterilizing protection was observed in one volunteer coimmunized with MSP1+AMA1—results consistent with vaccine-induced pre-erythrocytic, rather than blood-stage, immunity. These data call into question the utility of T cell-inducing blood-stage malaria vaccines and suggest that the focus should remain on high-titer antibody induction against susceptible antigen targets.

Introduction

Malaria continues to impose an unacceptable burden on global public health, with the most severe and life-threatening forms of the disease caused by Plasmodium falciparum.1 A highly effective vaccine, combined with pre-existing and partially effective control and elimination strategies could have a significant impact on malaria-associated morbidity and mortality. The most advanced malaria vaccine developed to date, RTS,S/AS01, is currently in Phase III clinical trials, with an interim report suggesting modest (30–50%) efficacy against clinical disease in young children.2 This protein-adjuvant vaccine, based on the circumsporozoite protein (CSP) carried by a recombinant hepatitis B surface antigen particle targets the pre-erythrocytic forms of the parasite. The subsequent blood-stage of infection is responsible for clinical illness and begins with the rupture of infected hepatocytes. This releases tens of thousands of merozoites into the circulation, ready to commence their repeated cycles of host RBC invasion followed by intraerythrocytic asexual replication. To date, most blood-stage subunit vaccine development efforts have focused on merozoite antigens formulated as recombinant protein-in-adjuvant, designed to induce functional antibodies capable of neutralizing parasite invasion of host RBCs (so-called growth inhibitory activity (GIA)), or activating Fc-receptor–bearing cells (antibody-dependent cellular inhibition).3,4 However, this strategy of targeting leading antigens such as merozoite surface protein 1 (MSP1),5 apical membrane antigen 1 (AMA1),6 and MSP3 have faced significant challenges, yielding limited success so far in clinical trials. Exceptionally high antibody titers appear to be required to confer protective efficacy,7,8 strong vaccine adjuvants that are safe and suitable for clinical use are often not readily accessible, and high levels of antigen polymorphism can render even the most promising vaccine-induced antibody responses strain-specific.9

Limited understanding of immune mechanisms that could confer protection in vivo in humans against blood-stage parasites has also confounded vaccine development. Studies in animal models of malaria and of naturally acquired immunity in human populations have suggested protective roles for multiple innate as well as adaptive cellular and humoral effectors. Indeed, it has been proposed that it may be essential to induce strong cellular immunity against target blood-stage antigens, in conjunction with functional antibodies, in order to achieve vaccine efficacy against this pathogenic stage of the parasite's life cycle.10 Strong CD4+ T-cell responses may provide essential help for B cells; may polarize induction of cytophilic IgG subclasses that better mediate antibody-dependent cellular inhibition via monocytes4 or antibody-dependent respiratory burst via neutrophils;11 or may activate splenic macrophages for improved opsonization of infected RBCs or production of proinflammatory parasiticidal cytokines.12 CD8+ T cells against blood-stage antigens may also target hepatocytes harboring developing merozoites at the late liver stage.13 However, to date, this hypothesis has not been investigated in human studies due to the absence of vaccine candidates capable of inducing strong cellular immunity against blood-stage antigens.

In recent years, we have described the development of blood-stage malaria viral-vectored vaccines delivered in the form of a recombinant adenovirus prime–poxvirus boost regimen. This approach has been shown to be highly immunogenic for both IgG antibody and T-cell induction in mice,14 rabbits,15,16 and rhesus macaques.17 In the P. yoelii rodent malaria model, this vaccination regimen induces high-titer MSP1-specific IgG antibodies that are effective against blood-stage parasites,14 as well as CD8+ T cells that target late liver-stage parasites.18 Liver-stage efficacy could also be enhanced in the P. yoelii model by coadministering viruses recombinant for the blood-stage antigen MSP1 with those encoding the pre-erythrocytic antigen CSP.19 Importantly, this same adenovirus-modified vaccinia virus Ankara (MVA) vaccination regimen, using vectors encoding AMA1, was also shown to mediate control of blood-stage P. chabaudi infection. In this alternative rodent malaria model, control of initial parasitemia was dependent on CD4+ T-cell responses acting in conjunction with antibodies.20 These data established a role, at least in mouse malaria models, for a protective contribution of both T cells and antibodies induced by adenovirus-MVA–based vaccines against blood-stage antigens.

More recently, we have shown this vaccine delivery platform to be highly immunogenic in healthy adults in Phase Ia clinical trials. The P. falciparum blood-stage antigens MSP1 or AMA1 were delivered by replication-defective chimpanzee adenovirus 63 (ChAd63) followed 8 weeks later by a boost with the attenuated orthopoxvirus MVA.21,22 Importantly, these vaccines were safe and induced exceptionally strong levels of antigen-specific CD4+ and CD8+ T cells, as well as substantial levels of antigen-specific IgG (average 40–60 µg/ml). These Phase Ia data are in agreement with other vaccine programmes (tested to date in humans and/or nonhuman primates) utilizing similar strategies to induce strong cellular immunity against the pre-erythrocytic stage malaria antigen multi-epitope string thrombospondin-related adhesion protein (ME-TRAP),23 hepatitis C virus,24 and HIV-1.25 However, to date, no vaccine programme has reported on efficacy of this platform against human disease. Here, we report the efficacy of these T cell- and antibody-inducing vaccines in healthy malaria-naive adults against controlled human malaria infection (CHMI) delivered by mosquitoes infected with 3D7-strain P. falciparum sporozoites in an initial small pilot safety Phase IIa study (VAC037) and a subsequent larger Phase IIa study (VAC039). The blood-stage malaria vaccines were administered alone (MSP1 versus AMA1), coadministered together (MSP1+AMA1) or coadministered with a pre-erythrocytic malaria vaccine candidate (MSP1+ME-TRAP).

Results

ChAd63-MVA MSP1 safety and efficacy following sporozoite challenge (VAC037)

Three volunteers vaccinated with 5 × 1010 vector particle ChAd63 followed 8 weeks later by 2 × 108 plaque-forming unit (pfu) MVA MSP1 underwent pilot sporozoite challenge with the 3D7 strain of P. falciparum 13–16 days following MVA MSP1 immunization. These three volunteers represent Group 2C from a previously reported Phase Ia study (VAC037) where safety and immunogenicity is described (Supplementary Figure S1a–c).21 This small initial CHMI study was conducted for safety reasons, because of a very small potential risk of inducing immunopathology upon challenge following the induction of high levels of effector T cells to a blood-stage antigen that are not found after natural exposure. There is evidence of such immunopathology in some murine models,26,27 although our experience in mice has been that strong cellular responses are, if anything, protective.18,20

Following mosquito bite CHMI, all nine volunteers (three MSP1 vaccinees and six infectivity controls) developed blood-stage malaria and no unexpected adverse events (AEs) occurred. The control volunteers were diagnosed after a median time of 11.4 days (range 9.0–12.3). There was a small though statistically significant delay in time to diagnosis by microscopy for the MSP1 vaccinees compared with control volunteers (P = 0.035) (Figure 1a), but no sterilizing protection. Blood-stage parasitemia was monitored twice daily by real-time quantitative PCR (qPCR) in each volunteer (Supplementary Figure S2a), with the median levels of parasitemia in each group consistent with the timing of diagnosis by microscopy (Figure 1b). The trial's primary outcome measure was the assessment of safety. Importantly, there was no significant difference between vaccinees and controls in terms of duration of symptoms before diagnosis (P = 0.89, Supplementary Figure S2b) or number of symptoms at diagnosis (P = 0.69, Supplementary Figure S2c). No unexpected AEs, clinical or laboratory signs of immunopathology were observed in vaccinees post-challenge.

Figure 1
Pilot safety Phase IIa study VAC037: efficacy of ChAd63-MVA MSP1 immunization following Plasmodium falciparum 3D7 strain sporozoite challenge. (a) Kaplan–Meier survival analysis of time to patent parasitemia in days (calculated from hours between ...

VAC039 study recruitment and vaccinations

In light of the results in the VAC037 Phase IIa safety study, recruitment took place between May 2010 and September 2010 for a larger Phase IIa study (VAC039). Thirty-eight healthy malaria-naive adult volunteers (21 female and 17 male) were enrolled across three sites in the UK. Six malaria-naive adult volunteers were enrolled to undergo CHMI as unvaccinated infectivity controls (two female and four male) (Figure 2). The mean age of volunteers was 29.5 years (range 19–50). Vaccinations began in July 2010, CHMI occurred in October 2010 and all follow-up visits were completed by March 2011. All vaccinees received their immunizations as scheduled. All doses of vaccines were the same as those used in the comparable Phase Ia studies,21,22 with the exception of MVA MSP1 where a reduced dose of 2 × 108 pfu was used to permit combined dosing (dose in Phase Ia study VAC037: 5 × 108 pfu). One volunteer in Group 1 was withdrawn from the study 10 days after MVA MSP1 (see below) but all other volunteers underwent CHMI as scheduled.

Figure 2
VAC039 flow chart of study design and volunteer recruitment. Eleven volunteers were excluded following screening for the following reasons: proteinuria, hypertension (two individuals), unexplained “jerking” episode, past medical history ...

Vaccine safety and reactogenicity

No unexpected or serious AEs related to vaccination occurred. The local (Supplementary Figure S3a,b) and systemic (Supplementary Figure S3c,d) reactogenicity profile of each vaccine administered alone was similar to Phase Ia data,21,22 with most AEs mild in severity. In the case of MVA MSP1, 2 × 108 pfu was associated with reduced local and systemic reactogenicity (Supplementary Figure S3b,d) in comparison to the dose of 5 × 108 pfu used in the Phase Ia study.21 While coadministration of vaccines in contralateral arms did not affect local reactogenicity (Supplementary Figure S3a,b) in comparison to that seen previously with each of the vaccines,21,22,23 it was associated with increased intensity of systemic reactogenicity consistent with the increased dose of vector administered. Coadministration of ChAd63-vectored vaccines (Figure 3a) appeared systemically more reactogenic than coadministration of MVA-vectored vaccines (Figure 3b). ChAd63 MSP1 + ChAd63 AMA1 (Group 3) appeared a more systemically reactogenic combination than ChAd63 MSP1 + ChAd63 ME-TRAP (Group 4). This was despite the total dose of ChAd63 (1 × 1011 vp) being the same for each group. MVA MSP1 + MVA ME-TRAP (total dose of MVA 4 × 108 pfu) appeared a slightly more systemically reactogenic combination than MVA MSP1 + MVA AMA1 (total dose of MVA 3.25 × 108 pfu) consistent with the increased dose of MVA vector administered in this vaccine combination. One volunteer in Group 1 developed appendicitis requiring surgery 10 days after MVA MSP1, was withdrawn from the study and did not undergo CHMI. This serious AE was deemed unlikely related to vaccination. No laboratory AEs were noted that were possibly, probably or definitely related to vaccination.

Figure 3
Systemic AEs deemed definitely, probably or possibly related to coadministration of ChAd63- or MVA-vectored vaccines (Groups 3 and 4). Only the highest intensity of each AE per subject is listed. Data are combined for all AEs for all volunteers receiving ...

ChAd63-MVA T-cell immunogenicity assessed by ex-vivo IFN-γ ELISPOT

The kinetic and magnitude of the vaccine-induced T-cell responses were assessed over time by ex-vivo interferon (IFN)-γ ELISPOT following restimulation of peripheral blood mononuclear cell (PBMC) with overlapping peptides spanning the MSP1, AMA1 or ME-TRAP inserts present in the viral-vectored vaccines (Figure 4a). In all cases, vaccination with ChAd63 vaccines primed antigen-specific IFN-γ–secreting T-cell responses that peaked 14–28 days later, before contraction and subsequent boosting by MVA administration at day 56. The responses observed in the single vaccine administration groups (Group 1 and Group 2) were consistent with those observed in the previous Phase Ia clinical trials of the MSP1 and AMA1 vaccines.21,22 This was despite a reduction in the dose of MVA MSP1 from 5 × 108 pfu in the previous VAC037 trial21 to 2 × 108 pfu in the VAC039 trial reported here (Supplementary Figure S1a). We also separately assessed responses against peptides spanning artificial junction sequences present within the vaccine inserts, and none were observed bar weak responses in six MSP1-immunized volunteers after the MVA boost, and one transient response in an AMA1-immunized volunteer at the day 28 timepoint (Supplementary Figure S4).

Figure 4
Cellular immunogenicity of ChAd63-MVA immunization regimens. (a) Median ex-vivo IFN-γ ELISPOT responses (summed response across all the individual peptide pools) in PBMCs are shown for each relevant Group to the MSP1, AMA1, ME-TRAP antigens. The ...

Interestingly, coadministration of the MSP1 vaccines with either those encoding AMA1 (Group 3) or ME-TRAP (Group 4), led to a significant reduction in MSP1-specific IFN-γ–secreting T-cell responses after the ChAd63 prime, and this was also observed after the MVA boost (Figure 4b). On the day before challenge (dC-1), the median MSP1-specific T-cell responses were reduced by 2.5-fold in Group 3 and 2.9-fold in Group 4 in comparison to Group 1. A similar effect was observed for AMA1-specific IFN-γ–secreting T-cell responses, which were significantly reduced after priming and boosting when the vaccines were coadministered with those encoding MSP1 (Group 3). On dC-1, the median AMA1-specific response was reduced by 4.7-fold in comparison to single vaccine administration in Group 2. Responses to ME-TRAP in Group 4 were of a similar magnitude to those seen for AMA1 in Group 3, and again were lower than those previously observed when the same vaccine regimen was administered alone in a Phase Ia trial.23 However, when the transgene-specific responses were summed across the two antigens in the coadministration groups, there was substantially less intergroup variation.

These data confirm the ability of this vaccine delivery platform to induce strong T-cell responses. They also show, however, that ChAd63-MVA MSP1 vaccine coadministration with the same vectors encoding AMA1 or ME-TRAP, does not increase the total antigen-specific T-cell response (despite contralateral-arm immunization). T-cell responses against the MSP1 antigen also appear to be immunodominant over those against the AMA1 and ME-TRAP antigens.

Antigen-specific T-cell phenotype

CD3+ T-cell phenotype was assayed at the dC-1 timepoint by restimulation of PBMCs with separate pools of peptides for each administered antigen, followed by intracellular cytokine staining (Figure 4c and Supplementary Figure S5). Similar to previous reports for all three antigens,21,22,23 the transgene-specific CD3+ T cells consisted of a mixed CD4+ and CD8+ phenotype. Once again, responses in the two single vaccine administration groups were very similar to those observed at the most comparable timepoint in the Phase Ia studies21,22 (Supplementary Figure S1c). CD4+ T cells produced IFN-γ, tumor necrosis factor-α, and interleukin-2 upon peptide restimulation, while CD8+ T cells expressed the cytotoxic T lymphocyte degranulation marker CD107a, as well as producing IFN-γ, tumor necrosis factor-α, and lower levels of interleukin-2. Very similar trends were observed with the ex-vivo ELISPOT assay in the two vaccine coadministration groups, with reduced responses observed to each antigen. Significant reductions were apparent for all responses against AMA1 following coadministration with MSP1, and similarly, responses to TRAP were low to undetectable (unlike those previously reported following single vaccine administration).23 As before, responses were split across the two malaria antigens expressed by the vaccines, but MSP1 appeared to be consistently immunodominant.

ChAd63-MVA antibody immunogenicity

The kinetic and magnitude of the vaccine-induced serum IgG antibody response were assessed over time by ELISA against the 3D7/MAD20 allele of MSP119 (Figure 5a) and the 3D7 allele of AMA1 (Figure 5b). In both cases, vaccination with ChAd63 vaccines primed antigen-specific IgG responses that in most cases peaked 28 days later and were maintained out to day 56, when they were boosted by MVA administration. The responses observed in the single vaccine administration groups were again consistent with those observed in the previous Phase Ia clinical trials, where these titers were shown on average to be equivalent to 40–60 µg/ml antigen-specific IgG21,22 (Supplementary Figure S1b). Unlike T-cell responses, IgG responses against MSP119 were not significantly reduced following coadministration of the ChAd63 MSP1 vector with those encoding AMA1 or ME-TRAP (Figure 5c). However, following the MVA boost, there was again a trend for reduced responses which reached significance in the case of Group 3. Thus on dC-1, the geomean MSP119-specific serum IgG responses were reduced by 2.4-fold in Group 3 and 1.8-fold in Group 4 in comparison to Group 1. In accordance with the T-cell data, antibody responses against MSP119 appeared to dominate over those against AMA1. The latter were significantly reduced both after priming and boosting when the vaccines were coadministered with those encoding MSP1 (Group 3). On dC-1, the geomean AMA1-specific response was reduced by 3.8-fold in comparison to single vaccine administration (Group 2).

Figure 5
IgG antibody responses and functional GIA induced by ChAd63-MVA immunization regimens. Geomean serum IgG ELISA responses are shown for all Groups to the 3D7 allele of the (a) MSP119 and (b) AMA1 antigens. (c) Individual and geomean responses are shown ...

In agreement with published data for the Phase Ia trials of ChAd63-MVA MSP121 and AMA1,22 human IgG responses of the magnitude measured here against MSP119 did not induce functional GIA above baseline against the 3D7 CHMI strain of P. falciparum in vitro (Figure 5d), while a subset of AMA1-immunized volunteers in Group 2 showed some modest functional activity. This level was comparable with that previously reported in volunteers boosted with the 1.25 × 108 pfu dose of MVA AMA1.22 Functional GIA was not improved by the simultaneous presence of lower level responses against the two blood-stage antigens (MSP1 and AMA1) in Group 3. Antibody responses against TRAP were not assessed and these would not be expected to induce functional activity against blood-stage parasites, although notably one volunteer in Group 4 showed a high GIA response.

ChAd63-MVA efficacy of all regimens following sporozoite challenge

Given the absence of safety concerns in the VAC037 pilot study, the larger Phase IIa study (VAC039) was undertaken. All volunteers in Groups 1–4 (bar one volunteer in Group 1), along with six unimmunized control volunteers (Group 5), underwent sporozoite challenge 14–18 days after the MVA boost immunization (Figure 2) (except two volunteers who, due to scheduling conflicts, were boosted earlier and thus challenged 24 (Group 3) and 25 (Group 4) days after MVA boost immunization). One volunteer in Group 1 withdrew 6 days after CHMI for reasons unrelated to the study and was not included in analysis of efficacy data. All the remaining 42 volunteers were monitored as before. Following mosquito bite CHMI, no unexpected AEs occurred. The infectivity controls (Group 5) and 35/36 vaccinees were diagnosed with malaria. One volunteer in Group 3 (AMA1+MSP1) was sterilely protected (Figure 6 and Supplementary Figure S6). The control volunteers (Group 5) were diagnosed after a median time of 9.7 days (range 8.9–11.2). In this study, there was no significant overall delay in MSP1-immunized individuals (Group 1) although one volunteer remained undiagnosed until dC + 12.8. A similar result was observed for Group 2 (AMA1), except for one volunteer who remained parasite negative by qPCR until dC + 14.5 and was finally diagnosed by microscopy on dC + 18.0. Following coimmunization with MSP1+AMA1 (Group 3), two volunteers were diagnosed on dC + 12.9 and dC + 13.3, while one volunteer was sterilely protected and did not show any detectable parasitemia by qPCR out to the final timepoint (dC + 21). However, overall efficacy in Group 3 did not reach statistical significance (P = 0.071). In the final group of volunteers coimmunized with MSP1+ME-TRAP, one remained parasite negative by qPCR until dC + 12.0, and was finally diagnosed by microscopy on dC + 15.0.

Figure 6
Efficacy of ChAd63-MVA immunization following Plasmodium falciparum 3D7 strain sporozoite challenge. Kaplan–Meier survival analysis of time to patent parasitemia in days (calculated from hours between mosquito bite and diagnosis) for the VAC039 ...

Modeling of parasitemia measured by qPCR

A delay to patency caused by a blood-stage vaccine could be due to reductions in either the liver-to-blood inoculum (LBI) and/or blood-stage parasite multiplication rate (PMR). In the case of both CHMI studies, there were no statistically significant overall differences observed between vaccinees and controls in either the LBI or PMR parameters as calculated using the sine-wave (Figure 7a,b), linear (Supplementary Figure S7) or cumulative density function (Supplementary Figure S9) models. We also observed a strong correlation between the outputs of the linear and sine-wave models (Supplementary Figure S8). It was noted that there was a trend in the second study for reduced median LBI in the vaccinated groups (Figure 7a), which mirrored analysis of the geomean qPCR data for each group at the early monitoring time points (at dC + 7 to dC + 8.5 just after the onset of blood-stage parasitemia) (Figure 7c). Such reduction in blood-stage inoculum would normally be associated with partial pre-erythrocytic vaccine efficacy as previously observed in the P. yoelii mouse model for similar viral vaccines encoding MSP1.18 In agreement with this, there was a strong correlation between time to diagnosis and LBI but not PMR (Figure 7d,e), confirming there was no effect on PMRs in this study and that delayed microscopic diagnosis was associated with a reduction in the estimated parasite load seeded from the liver into the blood. Those vaccinees showing a delayed time to diagnosis across all the groups exhibited low LBI, but not PMR (Figure 7d), suggesting that partial pre-erythrocytic efficacy rather than blood-stage immunity was observed. Nonetheless, this remained insufficient, in all bar one volunteer, to induce protective sterilizing immunity.

Figure 7
Sine-wave modelling of qPCR data. (a) Liver-to-blood inocula (LBI) and (b) parasite multiplication rates (PMR) as calculated using the model based upon a sine-wave.38 Values are indicated for both Phase IIa challenge studies (VAC039 to the left of the ...

Interestingly, a comparison of the two control groups used in the two different challenge studies showed a significantly higher LBI in the VAC039 compared with the VAC037 study (P = 0.02 for both sine-wave and linear models, Mann–Whitney test), but no significant difference in PMR. These data suggest the second challenge was more stringent, leading to a greater liver-stage parasite burden and subsequently a 36-fold increase in the median LBI (as estimated by the sine-wave model).

ChAd63-MVA safety of all regimens following sporozoite challenge

Six days following CHMI, a volunteer in Group 1 went missing from follow-up associated with a life event unrelated to the trial. He was next assessed clinically 18 days after CHMI when he had some mild symptoms of malaria and a positive blood film, and was treated successfully. This volunteer's data after CHMI are not included in analyses. Otherwise, no unexpected clinical or laboratory signs were observed in vaccinees post-challenge and in particular there was no evidence of any immunopathology. There was no difference between vaccinees and controls in the time that individuals were symptomatic before diagnosis (P = 0.92) or the number of symptoms present at time of diagnosis (P = 0.22) (Figure 8a,b). Eleven of the 41 volunteers (27%) diagnosed with malaria after CHMI had no symptoms of malaria infection at diagnosis (Figure 8b).

Figure 8
Analysis of CHMI clinical data. (a) Comparison of the number of symptomatic days before malaria diagnosis between vaccinees who underwent CHMI and were diagnosed with malaria (n = 35) and unvaccinated controls (n = 6) (P = 0.92, Mann–Whitney test). ...

The total duration of symptoms in volunteers with symptomatic malaria infection ranged from 1 to 18.5 days (median 4.8 days) with no significant difference in duration of symptoms following CHMI between vaccinees and controls (P = 0.58) (Figure 8c). Twenty-three volunteers (56%) experienced at least one AE post-challenge that was severe in intensity (Figure 8d). One volunteer in Group 5 was admitted for inpatient management of malaria symptoms 1 day post-malaria diagnosis, and was discharged the next day with no long-term sequelae. Safety bloods taken at dC + 9, dC + 35, dC + 90 and within 24 hours of diagnosis demonstrated transient abnormalities at frequencies and severities expected following P. falciparum infection28 (Figure 8e).

Discussion

The development of an efficacious vaccine against the blood stage of P. falciparum malaria has proven extremely challenging. It has been argued that the induction of cellular immunity, in conjunction with antibodies, may be essential for vaccines to confer protective efficacy against the blood-stage parasite.10 Such a strategy is supported by extensive data from the P. chabaudi rodent malaria model,20,27,29 as well as immunoepidemiological30 and experimental human studies.31 Here, we sought to assess for the first time in humans whether vaccine candidates that have been optimized for both antibody and T-cell induction against the MSP1 and AMA1 antigens15,16 could confer protective efficacy against blood-stage parasitemia in a CHMI study. We show that the induction of strong cellular immunity by viral-vectored vaccines encoding MSP1 and AMA1 does not impact on parasite growth rates in the blood of malaria-naive adults following mosquito bite CHMI. In a subset of vaccinated volunteers, sterilizing immunity or a delay in time to diagnosis without an altered PMR was observed; effects consistent with vaccine-induced pre-erythrocytic, rather than blood-stage, immunity.

In recent years, the adenovirus prime—poxvirus boost regimen has become widely recognized as a leading and versatile vaccine delivery platform capable of inducing both strong T-cell responses as well as moderate antibody responses in species ranging from mice to humans.14,17,21,22 This platform is now in advanced stages of development against a variety of difficult infectious disease targets including liver-stage malaria,23 hepatitis C virus,24 and HIV-1.25 However, no clinical study to date has assessed the safety or immunogenicity of coadministered vectors. Here, we report that the coadministration of two ChAd63 vectors (total dose 1 × 1011 vp) as well as two MVA vectors (highest total dose 4 × 108 pfu) displays an acceptable safety and reactogenicity profile. Similarly the more severe reactogenicity profile observed with higher doses of MVA MSP1 and AMA1 used in the Phase Ia clinical trials,21,22 was no longer apparent when the dose of MVA used was in the range of 1–2 × 108 pfu. Importantly, the magnitude of the observed T cell and IgG antibody responses was not reduced following the reduction of the dose of MVA MSP1 from 5 × 108 pfu to 2 × 108 pfu.

Previous studies in the P. yoelii mouse model using vectors encoding MSP1 and CSP had suggested that vaccine coadministration at separate sites could largely overcome immune interference between the two antigens.19 However, following vaccine coadministration here in humans, significantly reduced T cell and IgG antibody responses were observed, despite contralateral-arm vaccine administration. Notably, the MSP1 antigen was dominant over both AMA1 and ME-TRAP. Interestingly, nonhuman primate studies combining 42 kDa C-terminal region of MSP1 (MSP142) and AMA1 protein vaccines observed similar results, whereby coadministration of MSP142 and AMA1 protein vaccines led to a suppression of AMA1 but not MSP1-specific antibody responses, even when administered at separate sites.32,33 This could potentially reflect antigenic properties of this major surface protein of the merozoite—which may serve as an immune decoy mechanism, detracting from immune response induction against more protective targets. Conversely, a recently reported trial using two adenovirus human serotype 5 vectors encoding CSP and AMA1 administered ipsilaterally did not report immune interference—although the immune responses induced appeared substantially lower than those reported here.34 In many clinical trials, coadministered or multiantigen vaccines are not assessed alone and together making the interpretation of immune interference extremely difficult.35,36 Overall, the existing data suggest immune interference is complex and antigen-dependent. Consequently, any future multicomponent or multiantigen vaccine formulation needs to be carefully assessed, especially when using a delivery platform (such as adenovirus-MVA) that is highly immunogenic, as preclinical studies of vectored vaccines have indicated more marked antigenic interference with stronger immune responses.19 It will also be important to determine whether reduced immune responses against two antigens are more or less likely to confer protective efficacy than stronger responses against one.

In this study, we initially undertook a pilot safety trial in three volunteers immunized with the ChAd63-MVA MSP1 vaccines. This showed the vaccination approach to be safe following the development of blood-stage infection. Despite a small sample size, this pilot study showed a statistically significant delay in time to diagnosis in humans afforded by a vaccine targeting the MSP1 antigen alone. Although early vaccine-induced control of blood-stage parasitemia that is subsequently lost cannot be ruled out, trends for reduced LBI and delayed diagnosis in vaccinees (in the absence of reduced PMR or serum GIA) provided some preliminary evidence in humans that the classically termed P. falciparum blood-stage antigen MSP1 is infact a multistage antigen and can be targeted in the liver. These data concur with antigen expression data from chimpanzees,37 and challenge data in mice showing efficacy of MSP1-specific CD8+ T cells against liver-stage parasites.18 A median delay of 2.5 days to diagnosis would equate to >90% reduction in mean liver parasite burden,38 an effect larger than that reported in mice and possibly reflecting the 6–7 days, rather than 48 hours, liver-stage infection of humans and rodents, respectively.

Subsequently, the larger Phase Iia trial reported here failed to replicate this result in MSP1-immunized volunteers, and similarly no statistically significant efficacy was observed in any other group immunized with the vaccine candidates. This result is potentially influenced by the significantly larger parasite load apparent in the second study, despite the same standardized administration of five infectious mosquito bites. These data highlight the variability that can be observed between CHMI studies, despite extensive efforts to harmonize protocols and procedures. In agreement with the pilot study, there were no associated safety concerns, confirming that the induction of high levels of effector T cells to two different blood-stage antigens does not appear to induce immunopathology in humans, as found with some host–parasite combinations in murine models,26,27 at least during early experimental blood-stage exposure.

Encouragingly, one volunteer was sterilely protected—the first human fully protected against CHMI following vaccination with the AMA1 or MSP1 antigens. Similarly, a subset of vaccinees diagnosed late also showed low LBI, consistent with the pre-erythrocytic immune effect observed in the pilot study. In the absence of significant efficacy and with only one fully protected volunteer, immune association analyses were not performed, but it remains possible that such immunity could be mediated by anti-AMA1 antibodies against sporozoites39 and/or CD8+ T cell-mediated efficacy against infected hepatocytes.13,18 Because higher levels of efficacy have been observed when using the ChAd63-MVA ME-TRAP vaccines alone (K.J. Ewer, G.A. O'Hara, C.J.A. Duncan, K.A. Collins, S.H. Sheehy, A. Reyes-Sandoval et al., submitted), it would seem that TRAP remains a more promising target for liver-stage immunity, potentially due to a longer or earlier time of expression within the liver in comparison to merozoite-stage antigens40 and therefore greater opportunity for antigen presentation to effector cytotoxic T lymphocytes. Similarly the absence of efficacy in the MSP1+ME-TRAP group is in agreement with preclinical data showing reduced vaccine efficacy when protective responses are reduced due to immune interference.19

An important observation was that strong cellular immunity against two different blood-stage antigens did not impart any reduction in PMRs in humans. The contribution of cell-mediated efficacy against the blood-stage parasites has been strongly advocated,10 particularly in the widely used P. chabaudi rodent malaria model,20,27,29 as well as in human studies.30,31 It remains possible that the T cells induced here by vaccination were still of insufficient magnitude, or were of nonprotective functional phenotype or antigen-specificity.27 Nevertheless, the same viral-vectored vaccine approach using the P. chabaudi AMA1 antigen could induce CD4+ T cells that contributed to control of initial parasitemia in the mouse model.20 These data therefore call into question the utility for vaccine development of this particular preclinical model of cell-mediated blood-stage immunity. Other animal models, such as P. yoelii in rodents or nonhuman primate challenge systems, where extremely high-titer IgG responses are necessary for blood-stage protection,7,8,14 may more reliably reflect the ability of anti-merozoite vaccine candidates to induce protection in humans.

The antibody titers reported here were comparable to those observed in the Phase Ia trials,21,22 and in those studies we showed the ELISA titers equated on average to 40–60 µg/ml antigen-specific IgG. In the case of MSP1, although the IgG recognize native parasite antigen,21 titers of this magnitude are too low to mediate functional GIA in vitro. A previous study has shown roughly 600 µg/ml anti-MSP142 human IgG is required on average to achieve 50% GIA in vitro against 3D7 strain parasites.41 In the case of AMA1, ~100 µg/ml human IgG was required in the same study,41 in agreement with low level GIA observed here and the Phase Ia trial.22 CHMI by mosquito bite is also not optimal for detection of small vaccine-induced changes in PMR,42 and even the most promising protein AMA1 vaccine, achieving on average 200–280 µg/ml antigen-specific IgG in humans, did not achieve a measurable reduction in PMR following CHMI by mosquito bite.43 The absence of antibody efficacy in this trial would thus not be unexpected, given no apparent added protective contribution at the blood stage from the vaccine-induced T cells. We therefore conclude that future strategies should aim to (i) further enhance IgG induction by subunit vaccine delivery platforms, including the use of novel protein vaccine adjuvants as well as adenovirus prime—protein boost regimens,17,44 as well as (ii) target antigens that are susceptible to lower levels of vaccine-induced neutralizing antibodies, such as the recently described P. falciparum reticulocyte-binding protein homologue 5.45

We have described here the first coadministration of ChAd63-MVA–vectored vaccines against two antigens in humans, highlighting the importance of assessing immune interference in human studies. Moreover, the induction of strong cellular immunity against blood-stage malaria antigens was shown to be safe but incapable of impacting on initial parasite growth rates in the blood of malaria-naive adults following mosquito bite challenge. However, sterilizing immunity as well as a delay in time to diagnosis was observed, consistent with vaccine-induced pre-erythrocytic rather than blood-stage immunity. These data call into question the utility of T cell-inducing blood-stage malaria vaccine candidates with currently achievable levels of cellular immunogenicity, and suggest that the main focus for vaccines targeting blood-stage infection should remain on high-titer antibody induction against susceptible antigen targets.

Materials and Methods

Participants. The VAC039 study was conducted at the Oxford Vaccine Centre, part of the Centre for Clinical Vaccinology and Tropical Medicine, University of Oxford, UK; the University College London Clinical Research Facility, London, UK; and the NIHR Wellcome Trust Clinical Research Facility, University of Southampton and University Hospital Southampton NHS Foundation Trust, Southampton, UK. The challenge procedure was performed as previously described46 using five infectious bites from P. falciparum 3D7-strain infected Anopheles stephensi mosquitoes at the Alexander Fleming Building, Imperial College, London, UK, supplied by Jittawadee R Murphy, Department of Entomology, Walter Reed Army Institute of Research, Washington, DC, USA. Healthy, malaria-naive males and nonpregnant females aged 18–50 years were invited to participate in the study. All volunteers gave written informed consent before participation, and the study was conducted according to the principles of the Declaration of Helsinki and in accordance with Good Clinical Practice. There was no selection of volunteers on the basis of pre-existing neutralizing antibodies to the ChAd63 vector before enrolment (see Supplementary Materials and Methods for the full list of inclusion and exclusion criteria). All corresponding information relating to the pilot safety Phase IIa study conducted as part of a separate vaccine trial (VAC037, see ref. 21) can be found in Supplementary Materials and Methods.

Ethical and regulatory approval. All necessary approvals for VAC039 were granted by the Berkshire Research Ethics Committee (Ref: 10/H0505/30) and the UK Medicines and Healthcare products Regulatory Agency (Ref: 21584/0263/001-0001). Vaccine use was authorized by the Genetically Modified Organisms Safety Committee of the Oxford University Hospitals NHS Trust (Reference no. GM462.10.50). The trial was registered with ClinicalTrials.gov (Ref: NCT01142765). The Local Safety Committee provided safety oversight and Good Clinical Practice compliance was independently monitored by an external organization (Appledown Clinical Research, Great Missenden, UK).

ChAd63 and MVA vaccines. Generation, manufacture, and QC monitoring of the recombinant ChAd63 and MVA vectors encoding MSP1, AMA1, and ME-TRAP has been previously described.21,22,23 Briefly, the AMA1 transgene insert contains from N- to C-terminus: the leader sequence from human tissue plasminogen activator followed in-frame by sequence encoding the ectodomain of P. falciparum (strain 3D7) AMA1 linked to the ectodomain plus C-terminal transmembrane region of P. falciparum (strain FVO) AMA1.15,17,22 The MSP1 antigen (previously termed PfM128) was composed of the conserved blocks of sequence (1, 3, 5, and 12) from P. falciparum MSP1 followed by both of the known allelic sequences encoding the 42 kDa C-terminus fused in tandem.16,21 The antigen ME-TRAP contains a fusion protein of a ME followed by the P. falciparum T9/96 strain pre-erythrocytic TRAP.23

Study design. This was a Phase I/IIa open-label, nonrandomized vaccine and CHMI trial. Allocation to study groups (Figure 2) occurred at screening based on volunteer preference, as previously described.21 All vaccinations were administered intramuscularly into the deltoid. Details of dosing, clinical follow-up, and safety monitoring are given in Supplementary Materials and Methods and Supplementary Tables S4–S6. All the volunteers underwent sporozoite CHMI administered by mosquito bite as described in the results. A time window ranging between 1 and 14 days was allowed for vaccination and follow-up visits post-vaccination. Throughout the paper, study day refers to the nominal timepoint for a group and not the actual day of sampling.

PBMC and serum preparation. Blood samples were collected and PBMC and serum samples were isolated as previously described.21

Peptides for T-cell assays. Peptides were used to assess T-cell responses as previously described for each antigen,21,22,23 with some minor modifications (detailed in Supplementary Materials and Methods and Supplementary Table S1).

Ex-vivo IFN-γ ELISPOT. The T-cell response to the vaccine antigens were assessed over time by ex-vivo IFN-γ ELISPOT following an 18–20 hours restimulation of PBMC with overlapping peptides spanning the antigens present in the viral-vectored vaccines. Fresh PBMC were used in all ELISPOT assays using a previously described protocol.21,23 Spots were counted using an ELISPOT counter (Autoimmun Diagnostika, Strassberg, Germany). Results are expressed as IFN-γ spot-forming units per million PBMC. Background responses in unstimulated control wells were almost always <20 spots per 250,000 cells, and were subtracted from those measured in peptide-stimulated wells. The “Junctional” responses (shown in Supplementary Figure S4 and Supplementary Table S1), represent data measured using single pools of peptides.

Multiparameter flow cytometry. Cytokine secretion by PBMC was assayed by intracellular cytokine staining followed by flow cytometry using a previously described protocol.21 Briefly, frozen PBMC were restimulated for 18 hours in the presence of anti-human CD49d and CD28 (BD Biosciences, Oxford, UK) and CD107a. Restimulation for the final 16 hours was carried out in the presence of Brefeldin A (Sigma, Poole, UK) and Monensin (Golgi Stop; BD Biosciences). Each sample was restimulated with either: 1 µg/ml SEB (positive-control samples); the relevant pool(s) of antigen-specific peptides (see above) at final concentration 2 µg/ml each peptide and maximum 0.20% total DMSO concentration; and 0.20% DMSO final concentration (unstimulated peptide control sample). Cells were stained the next day using a live/dead marker, as well as for CD4, CD14, CD20, CD8α, CD3, IFN-γ, tumor necrosis factor-α, and interleukin-2. Samples were analyzed using a LSRII Flow Cytometer (BD Biosciences) and FlowJo v8.8 (TreeStar, Ashland, OR). Dead cells, monocytes (CD14+), and B cells (CD20+) were excluded from the analysis (Supplementary Figure S5). Background responses in unstimulated no peptide control cells were subtracted from the antigen-specific peptide responses.

Total IgG ELISA. ELISAs were performed against 3D7 AMA1 protein and ETSR (3D7 allele) MSP119 antigen using the same standardized methodology, as previously described.21,22

In vitro assay of GIA. The ability of vaccine-induced antibodies to inhibit growth of P. falciparum 3D7 strain parasites was assessed by a standardized GIA assay using purified IgG as previously described.41 Briefly, each test IgG (10 mg/ml in a final test well) was incubated with synchronized P. falciparum parasites for a single growth cycle and relative parasitemia levels were quantified by biochemical determination of parasite lactate dehydrogenase.

Parasite qPCR. Blood was collected and prepared for PCR as previously described.47 qPCR was performed in triplicate wells as previously described,48 except that (i) 5 µl extracted DNA template was used; (ii) the TaqMan probe: 5′ FAM-AAC AAT TGG AGG GCA AG-NFQ-MGB 3′ (Applied Biosystems, Foster City, CA) was used49; and (iii) 12.5 µl Universal PCR Master Mix was used on an Applied Biosystems Step One Plus PCR System with quantification performed by Applied Biosystems Step One plus software v2.1. Mean parasite equivalent values below 20 parasites/ml or with only one positive replicate of three tested were classed as negative.

Malaria diagnosis. For VAC037 diagnosis of malaria infection following sporozoite challenge was performed as previously described46 and defined as positive thick film microscopy with at least one morphologically normal malaria trophozoite seen by one or more experienced microscopists. Real-time qPCR for P. falciparum was simultaneously performed, although investigators directly involved in clinical management were blinded to these results. If a volunteer described symptoms or displayed signs which were likely to represent malaria infection in the opinion of the clinical investigators (such as fever, rigors or severe symptomatology), despite having a negative thick film and in the absence of an alternative cause, they were unblinded to the qPCR result. If this was positive the volunteer was treated for malaria. Time to diagnosis was measured in hours and converted to days for analysis.

For VAC039 diagnosis of malaria infection was performed as for VAC037 with the exception that for volunteers with positive thick film microscopy, but no symptoms consistent with P. falciparum malaria infection, investigators were unblinded to the qPCR results and the volunteer only treated if any preceding samples had >500 parasites/ml. This was to help reduce false-positive diagnosis by microscopy and to allow for the possibility that crisis forms46 may have been seen on microscopy in volunteers' controlling parasitemia.

Parasite growth modeling. The number of infected erythrocytes in the first generation after parasite release from the liver (LBI) and the PMR in the blood were estimated using qPCR data by application of three mathematical models; a sine-wave based model fitted to individuals as described by Bejon et al.38 (Figure 7), a model based upon the normal cumulative density function as described by Hermsen et al.50 (Supplementary Figure S9), and a linear model fitted to individual volunteers' qPCR data (A.D. Douglas, N.J. Edwards, C.J.A. Duncan, F. Thompson, S.H. Sheehy, G.A. O'Hara et al., manuscript in preparation) (Supplementary Figure S7). We analyzed the VAC037 data using all three models, but only applied the sine-wave and linear models to the VAC039 dataset. Models were fitted using Stata release 11 (Stata, College Station, TX). A more detailed comparison of these modeling methods is described elsewhere (A.D. Douglas, N.J. Edwards, C.J.A. Duncan, F. Thompson, S.H. Sheehy, G.A. O'Hara et al., manuscript in preparation). Full modeling details and methodology are provided in Supplementary Materials and Methods, with specific information pertaining to the separate VAC037 and VAC039 challenge outcomes.

Statistical analysis. Data were analyzed using GraphPad Prism version 5.03 for Windows (GraphPad Software, San Diego, CA). Individual, geometric mean or median responses for measurements within each group are described with the exception of intracellular cytokine staining data where box and whisker plots show the range, median, and interquartile range. Significance testing of differences between groups used the two-tailed Mann–Whitney U-test (two groups) or Kruskal–Wallis test with Dunn's multiple comparison test (more than two groups). Correlations were analyzed using Spearman's rank correlation coefficient. Significance testing by Kaplan–Meier survival analysis used the log-rank test. A value of P < 0.05 was considered significant.

SUPPLEMENTARY MATERIAL
Figure S1. Comparison of immune responses in ChAd63-MVA MSP1-immunized volunteers from VAC037 and VAC039.
Figure S2. VAC037 pilot challenge study—qPCR and safety data.
Figure S3. Local and systemic AEs deemed definitely, probably or possibly related to individual study vaccines administered in VAC039.
Figure S4. Junctional peptide responses.
Figure S5. Gating strategy for the analysis of antigen-specific T-cell responses.
Figure S6. VAC039 individual qPCR data.
Figure S7. Linear modeling of qPCR data.
Figure S8. Correlation of modeling outputs.
Figure S9. CDF modeling of VAC037 qPCR data.
Table S1. Alternative peptide sequences used in the VAC039 study.
Table S2. Raw qPCR data (parasites/ml) for the VAC037 Phase IIa safety study.
Table S3. Raw qPCR data (parasites/ml) for the VAC039 Phase IIa study.
Table S4. Assessment of relationship of AE to study intervention.
Table S5. Severity grading criterion for quantifiable AEs.
Table S6. Severity grading criterion of AEs.
Materials and Methods.

Acknowledgments

We thank Cynthia Bateman, Mary Smith, Joel Meyer, Raquel Lopez-Ramon, Roldan Singzon, Jung Ryu, Clare Grocott, and Filipa Martins for clinical assistance; Laura Dinsmore for logistical support; Simon Correa, Kebba Konteh, Peter Kalume, and Wycliffe Asava for microscopy expertise; Jittawadee Murphy, Ken Baker, Andrew Blagborough, Mark Tunnicliff, Brooke Bozick, Melissa Kapulu, and Arturo Reyes-Sandoval for assistance with the controlled human malaria infection studies; Julie Furze, Alexandra Spencer, Andrew Williams, Simon de Cassan, Emily Forbes, and Drew Worth for laboratory assistance; Philip Bejon and Fiona Thompson for advice on modeling parasite growth rates; and Laura Andrews, Chris Schultz, Jake Matthews, Aisling Vaughan, Matthew Dicks, and Fionnadh Carroll for assistance in quantitative PCR; the Jenner Institute Flow Cytometry Core Facility for technical assistance; Sam Moretz, Ababacar Diouf, and Gregory Tullo for technical support performing the growth inhibitory activity assays; and all the study volunteers. This work was supported by the European Malaria Vaccine Development Association, a European Commission FP6-funded consortium (LSHP-CT-2007-037506); the UK Medical Research Council (grant no. G0700735); the UK National Institute of Health Research through the Oxford Biomedical Research Centre (A91301 Adult Vaccine), and the Southampton NIHR Wellcome Trust Clinical Research Facility; the Wellcome Trust (084113/Z/07/Z); and EVIMalaR, an European Commission FP7-funded programme (grant agreement no. 242095). The growth inhibitory activity work was supported by the PATH Malaria Vaccine Initiative and the Intramural Program of the National Institutes of Health, National Institute of Allergy and Infectious Diseases. S.C.G., A.V.S.H., and S.J.D. are Jenner Investigators; A.V.S.H. was supported by a Wellcome Trust Principal Research Fellowship (45488/Z/05); C.J.A.D. holds a Wellcome Trust Research Training Fellowship (094449/Z/10/Z); and S.J.D. is a UK Medical Research Council Career Development Fellow (G1000527). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. A.D.D., S.C.G., A.V.S.H., and S.J.D. are named inventors on patent applications covering 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. The others authors declared no conflict of interest.

Supplementary Material

Figure S1.

Comparison of immune responses in ChAd63-MVA MSP1-immunized volunteers from VAC037 and VAC039.

Figure S2.

VAC037 pilot challenge study—qPCR and safety data.

Figure S3.

Local and systemic AEs deemed definitely, probably or possibly related to individual study vaccines administered in VAC039.

Figure S4.

Junctional peptide responses.

Figure S5.

Gating strategy for the analysis of antigen-specific T-cell responses.

Figure S6.

VAC039 individual qPCR data.

Figure S7.

Linear modeling of qPCR data.

Figure S8.

Correlation of modeling outputs.

Figure S9.

CDF modeling of VAC037 qPCR data.

Table S1.

Alternative peptide sequences used in the VAC039 study.

Table S2.

Raw qPCR data (parasites/ml) for the VAC037 Phase IIa safety study.

Table S3.

Raw qPCR data (parasites/ml) for the VAC039 Phase IIa study.

Table S4.

Assessment of relationship of AE to study intervention.

Table S5.

Severity grading criterion for quantifiable AEs.

Table S6.

Severity grading criterion of AEs.

Materials and Methods.

REFERENCES

  • Murray CJ, Rosenfeld LC, Lim SS, Andrews KG, Foreman KJ, Haring D. et al. (2012. Global malaria mortality between 1980 and 2010: a systematic analysis Lancet 379413–431.431 [PubMed]
  • Agnandji ST, Lell B, Soulanoudjingar SS, Fernandes JF, Abossolo BP, Conzelmann C. et al. (2011. First results of phase 3 trial of RTS,S/AS01 malaria vaccine in African children N Engl J Med 3651863–1875.1875 [PubMed]
  • Duncan CJ, Hill AV, andEllis RD. Can growth inhibition assays (GIA) predict blood-stage malaria vaccine efficacy. Hum Vaccin Immunother. 2012;8:706–714. [PMC free article] [PubMed]
  • Bouharoun-Tayoun H, Attanath P, Sabchareon A, Chongsuphajaisiddhi T, andDruilhe P. Antibodies that protect humans against Plasmodium falciparum blood stages do not on their own inhibit parasite growth and invasion in vitro, but act in cooperation with monocytes. J Exp Med. 1990;172:1633–1641. [PMC free article] [PubMed]
  • Holder AA. The carboxy-terminus of merozoite surface protein 1: structure, specific antibodies and immunity to malaria. Parasitology. 2009;136:1445–1456. [PubMed]
  • Remarque EJ, Faber BW, Kocken CH, andThomas AW. Apical membrane antigen 1: a malaria vaccine candidate in review. Trends Parasitol. 2008;24:74–84. [PubMed]
  • Dutta S, Sullivan JS, Grady KK, Haynes JD, Komisar J, Batchelor AH. et al. (2009. High antibody titer against apical membrane antigen-1 is required to protect against malaria in the Aotus model PLoS ONE 4e8138. [PMC free article] [PubMed]
  • Lyon JA, Angov E, Fay MP, Sullivan JS, Girourd AS, Robinson SJ. et al. (2008. Protection induced by Plasmodium falciparum MSP1(42) is strain-specific, antigen and adjuvant dependent, and correlates with antibody responses PLoS ONE 3e2830. [PMC free article] [PubMed]
  • Thera MA, Doumbo OK, Coulibaly D, Laurens MB, Ouattara A, Kone AK. et al. (2011. A field trial to assess a blood-stage malaria vaccine N Engl J Med 3651004–1013.1013 [PMC free article] [PubMed]
  • Good MF, andEngwerda C. Defying malaria: Arming T cells to halt malaria. Nat Med. 2011;17:49–51. [PubMed]
  • Joos C, Marrama L, Polson HE, Corre S, Diatta AM, Diouf B. et al. (2010. Clinical protection from falciparum malaria correlates with neutrophil respiratory bursts induced by merozoites opsonized with human serum antibodies PLoS ONE 5e9871. [PMC free article] [PubMed]
  • Walther M, Woodruff J, Edele F, Jeffries D, Tongren JE, King E. et al. (2006. Innate immune responses to human malaria: heterogeneous cytokine responses to blood-stage Plasmodium falciparum correlate with parasitological and clinical outcomes J Immunol 1775736–5745.5745 [PubMed]
  • Belnoue E, Voza T, Costa FT, Grüner AC, Mauduit M, Rosa DS. et al. (2008. Vaccination with live Plasmodium yoelii blood stage parasites under chloroquine cover induces cross-stage immunity against malaria liver stage J Immunol 1818552–8558.8558 [PubMed]
  • Draper SJ, Moore AC, Goodman AL, Long CA, Holder AA, Gilbert SC. et al. (2008. Effective induction of high-titer antibodies by viral vector vaccines Nat Med 14819–821.821 [PubMed]
  • Biswas S, Dicks MDJ, Long CA, Remarque EJ, Siani L, Colloca S. et al. (2011. Transgene optimization, immunogenicity and in vitro efficacy of viral vectored vaccines expressing two alleles of Plasmodium falciparum AMA1 PLoS One 6e20977. [PMC free article] [PubMed]
  • Goodman AL, Epp C, Moss D, Holder AA, Wilson JM, Gao GP. et al. (2010. New candidate vaccines against blood-stage Plasmodium falciparum malaria: prime-boost immunization regimens incorporating human and simian adenoviral vectors and poxviral vectors expressing an optimized antigen based on merozoite surface protein 1 Infect Immun 784601–4612.4612 [PMC free article] [PubMed]
  • Draper SJ, Biswas S, Spencer AJ, Remarque EJ, Capone S, Naddeo M. et al. (2010. Enhancing blood-stage malaria subunit vaccine immunogenicity in rhesus macaques by combining adenovirus, poxvirus, and protein-in-adjuvant vaccines J Immunol 1857583–7595.7595 [PubMed]
  • Draper SJ, Goodman AL, Biswas S, Forbes EK, Moore AC, Gilbert SC. et al. (2009. Recombinant viral vaccines expressing merozoite surface protein-1 induce antibody- and T cell-mediated multistage protection against malaria Cell Host Microbe 595–105.105 [PMC free article] [PubMed]
  • Forbes EK, Biswas S, Collins KA, Gilbert SC, Hill AV, andDraper SJ. Combining liver- and blood-stage malaria viral-vectored vaccines: investigating mechanisms of CD8+ T cell interference. J Immunol. 2011;187:3738–3750. [PMC free article] [PubMed]
  • Biswas S, Spencer AJ, Forbes EK, Gilbert SC, Holder AA, Hill AV. et al. (2012. Recombinant viral-vectored vaccines expressing Plasmodium chabaudi AS apical membrane antigen 1: mechanisms of vaccine-induced blood-stage protection J Immunol 1885041–5053.5053 [PMC free article] [PubMed]
  • Sheehy SH, Duncan CJ, Elias SC, Collins KA, Ewer KJ, Spencer AJ. et al. (2011. Phase Ia clinical evaluation of the Plasmodium falciparum blood-stage antigen MSP1 in ChAd63 and MVA vaccine vectors Mol Ther 192269–2276.2276 [PMC free article] [PubMed]
  • Sheehy SH, Duncan CJ, Elias SC, Biswas S, Collins KA, O'Hara GA. et al. (2012. Phase Ia clinical evaluation of the safety and immunogenicity of the Plasmodium falciparum blood-stage antigen AMA1 in ChAd63 and MVA vaccine vectors PLoS ONE 7e31208. [PMC free article] [PubMed]
  • O'Hara GA, Duncan CJ, Ewer KJ, Collins KA, Elias SC, Halstead FD. et al. (2012. Clinical assessment of a recombinant simian adenovirus ChAd63: a potent new vaccine vector J Infect Dis 205772–781.781 [PMC free article] [PubMed]
  • Barnes E, Folgori A, Capone S, Swadling L, Aston S, Kurioka A. et al. (2012. Novel adenovirus-based vaccines induce broad and sustained T cell responses to HCV in man Sci Transl Med 4115ra1 [PMC free article] [PubMed]
  • Barouch DH, Liu J, Li H, Maxfield LF, Abbink P, Lynch DM. et al. (2012. Vaccine protection against acquisition of neutralization-resistant SIV challenges in rhesus monkeys Nature 48289–93.93 [PMC free article] [PubMed]
  • Good MF, Xu H, Wykes M, andEngwerda CR. Development and regulation of cell-mediated immune responses to the blood stages of malaria: implications for vaccine research. Annu Rev Immunol. 2005;23:69–99. [PubMed]
  • Makobongo MO, Riding G, Xu H, Hirunpetcharat C, Keough D, de Jersey J. et al. (2003. The purine salvage enzyme hypoxanthine guanine xanthine phosphoribosyl transferase is a major target antigen for cell-mediated immunity to malaria Proc Natl Acad Sci USA 1002628–2633.2633 [PMC free article] [PubMed]
  • Epstein JE, Rao S, Williams F, Freilich D, Luke T, Sedegah M. et al. (2007. Safety and clinical outcome of experimental challenge of human volunteers with Plasmodium falciparum-infected mosquitoes: an update J Infect Dis 196145–154.154 [PubMed]
  • Xu H, Hodder AN, Yan H, Crewther PE, Anders RF, andGood MF. CD4+ T cells acting independently of antibody contribute to protective immunity to Plasmodium chabaudi infection after apical membrane antigen 1 immunization. J Immunol. 2000;165:389–396. [PubMed]
  • Riley EM, Allen SJ, Wheeler JG, Blackman MJ, Bennett S, Takacs B. et al. (1992. Naturally acquired cellular and humoral immune responses to the major merozoite surface antigen (PfMSP1) of Plasmodium falciparum are associated with reduced malaria morbidity Parasite Immunol 14321–337.337 [PubMed]
  • Pombo DJ, Lawrence G, Hirunpetcharat C, Rzepczyk C, Bryden M, Cloonan N. et al. (2002. Immunity to malaria after administration of ultra-low doses of red cells infected with Plasmodium falciparum Lancet 360610–617.617 [PubMed]
  • Pichyangkul S, Tongtawe P, Kum-Arb U, Yongvanitchit K, Gettayacamin M, Hollingdale MR. et al. (2009. Evaluation of the safety and immunogenicity of Plasmodium falciparum apical membrane antigen 1, merozoite surface protein 1 or RTS,S vaccines with adjuvant system AS02A administered alone or concurrently in rhesus monkeys Vaccine 28452–462.462 [PubMed]
  • Stowers AW, Kennedy MC, Keegan BP, Saul A, Long CA, andMiller LH. Vaccination of monkeys with recombinant Plasmodium falciparum apical membrane antigen 1 confers protection against blood-stage malaria. Infect Immun. 2002;70:6961–6967. [PMC free article] [PubMed]
  • Sedegah M, Tamminga C, McGrath S, House B, Ganeshan H, Lejano J. et al. (2011. Adenovirus 5-vectored P. falciparum vaccine expressing CSP and AMA1. Part A: safety and immunogenicity in seronegative adults PLoS ONE 6e24586. [PMC free article] [PubMed]
  • Saul A, Lawrence G, Smillie A, Rzepczyk CM, Reed C, Taylor D. et al. (1999. Human phase I vaccine trials of 3 recombinant asexual stage malaria antigens with Montanide ISA720 adjuvant Vaccine 173145–3159.3159 [PubMed]
  • Porter DW, Thompson FM, Berthoud TK, Hutchings CL, Andrews L, Biswas S. et al. (2011. A human Phase I/IIa malaria challenge trial of a polyprotein malaria vaccine Vaccine 297514–7522.7522 [PMC free article] [PubMed]
  • Szarfman A, Walliker D, McBride JS, Lyon JA, Quakyi IA, andCarter R. Allelic forms of gp195, a major blood-stage antigen of Plasmodium falciparum, are expressed in liver stages. J Exp Med. 1988;167:231–236. [PMC free article] [PubMed]
  • Bejon P, Andrews L, Andersen RF, Dunachie S, Webster D, Walther M. et al. (2005. Calculation of liver-to-blood inocula, parasite growth rates, and preerythrocytic vaccine efficacy, from serial quantitative polymerase chain reaction studies of volunteers challenged with malaria sporozoites J Infect Dis 191619–626.626 [PubMed]
  • Silvie O, Franetich JF, Charrin S, Mueller MS, Siau A, Bodescot M. et al. (2004. A role for apical membrane antigen 1 during invasion of hepatocytes by Plasmodium falciparum sporozoites J Biol Chem 2799490–9496.9496 [PubMed]
  • Bodescot M, Silvie O, Siau A, Refour P, Pino P, Franetich JF. et al. (2004. Transcription status of vaccine candidate genes of Plasmodium falciparum during the hepatic phase of its life cycle Parasitol Res 92449–452.452 [PubMed]
  • Miura K, Zhou H, Diouf A, Moretz SE, Fay MP, Miller LH. et al. (2009. Anti-apical-membrane-antigen-1 antibody is more effective than anti-42-kilodalton-merozoite-surface-protein-1 antibody in inhibiting plasmodium falciparum growth, as determined by the in vitro growth inhibition assay Clin Vaccine Immunol 16963–968.968 [PMC free article] [PubMed]
  • Duncan CJ, andDraper SJ. Controlled human blood stage malaria infection: current status and potential applications. Am J Trop Med Hyg. 2012;86:561–565. [PMC free article] [PubMed]
  • Spring MD, Cummings JF, Ockenhouse CF, Dutta S, Reidler R, Angov E. et al. (2009. Phase 1/2a study of the malaria vaccine candidate apical membrane antigen-1 (AMA-1) administered in adjuvant system AS01B or AS02A PLoS ONE 4e5254. [PMC free article] [PubMed]
  • de Cassan SC, Forbes EK, Douglas AD, Milicic A, Singh B, Gupta P. et al. (2011. 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 1872602–2616.2616 [PMC free article] [PubMed]
  • Douglas AD, Williams AR, Illingworth JJ, Kamuyu G, Biswas S, Goodman AL. et al. (2011. The blood-stage malaria antigen PfRH5 is susceptible to vaccine-inducible cross-strain neutralizing antibody Nat Commun 2601. [PMC free article] [PubMed]
  • Thompson FM, Porter DW, Okitsu SL, Westerfeld N, Vogel D, Todryk S. et al. (2008. Evidence of blood stage efficacy with a virosomal malaria vaccine in a phase IIa clinical trial PLoS ONE 3e1493. [PMC free article] [PubMed]
  • Andrews L, Andersen RF, Webster D, Dunachie S, Walther RM, Bejon P. et al. (2005. Quantitative real-time polymerase chain reaction for malaria diagnosis and its use in malaria vaccine clinical trials Am J Trop Med Hyg 73191–198.198 [PubMed]
  • Hermsen CC, Telgt DS, Linders EH, van de Locht LA, Eling WM, Mensink EJ. et al. (2001. Detection of Plasmodium falciparum malaria parasites in vivo by real-time quantitative PCR Mol Biochem Parasitol 118247–251.251 [PubMed]
  • Wang CW, Hermsen CC, Sauerwein RW, Arnot DE, Theander TG, andLavstsen T. The Plasmodium falciparum var gene transcription strategy at the onset of blood stage infection in a human volunteer. Parasitol Int. 2009;58:478–480. [PubMed]
  • Hermsen CC, de Vlas SJ, van Gemert GJ, Telgt DS, Verhage DF, andSauerwein RW. Testing vaccines in human experimental malaria: statistical analysis of parasitemia measured by a quantitative real-time polymerase chain reaction. Am J Trop Med Hyg. 2004;71:196–201. [PubMed]

Articles from Molecular Therapy are provided here courtesy of The American Society of Gene & Cell Therapy
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

    Your browsing activity is empty.

    Activity recording is turned off.

    Turn recording back on

    See more...