Immunoglobulin G subclass and antibody avidity responses in Malian children immunized with Plasmodium falciparum apical membrane antigen 1 vaccine candidate FMP2.1/AS02A.

BACKGROUND: A malaria vaccine based on Plasmodium falciparum apical membrane antigen 1 (AMA1) elicited strain specific efficacy in Malian children that waned in the second season after vaccination despite sustained AMA1 antibody titers. With the goal of identifying a humoral correlate of vaccine-induced protection, pre- and post-vaccination sera from children vaccinated with the AMA1 vaccine and from a control group that received a rabies vaccine were tested for AMA1-specific immunoglobulin G (IgG) subclasses (IgG1, IgG2, IgG3, and IgG4) and for antibody avidity. METHODS: Samples from a previously completed Phase 2 AMA1 vaccine trial in children residing in Mali, West Africa were used to determine AMA1-specific IgG subclass antibody titers and avidity by ELISA. Cox proportional hazards models were used to assess correlation between IgG subclass antibody titers and risk of time to first or only clinical malaria episode and risk of multiple episodes. Asexual P. falciparum parasite density measured for each child as area under the curve were used to assess correlation between IgG subclass antibody titers and parasite burden. RESULTS: AMA1 vaccination did not elicit a change in antibody avidity; however, AMA1 vaccinees had a robust IgG subclass response that persisted over the malaria transmission season. AMA1-specific IgG subclass responses were not associated with decreased risk of subsequent clinical malaria. For the AMA1 vaccine group, IgG3 levels at study day 90 correlated with high parasite burden during days 90-240. In the control group, AMA1-specific IgG subclass rise and persistence over the malaria season was modest and correlated with age. In the control group, titers of several IgG subclasses at days 90 and 240 correlated with parasite burden over the first 90 study days, and IgG3 at day 240 correlated with parasite burden during days 90-240. CONCLUSIONS: Neither IgG subclass nor avidity was associated with the modest, strain-specific efficacy elicited by this blood stage malaria vaccine. Although a correlate of protection was not identified, correlations between subclass titers and age, and correlations between IgG subclass titers and parasite burden, defined by area under the curve parasitaemia levels, were observed, which expand knowledge about IgG subclass responses. IgG3, known to have the shortest half-life of the IgG subclasses, might be the most temporally relevant indicator of ongoing malaria exposure when examining antibody responses to AMA1.

Demonstration of the Blood-Stage Plasmodium falciparum Controlled Human Malaria Infection Model to Assess Efficacy of the P. falciparum Apical Membrane Antigen 1 Vaccine, FMP2.1/AS01.

BACKGROUND: Models of controlled human malaria infection (CHMI) initiated by mosquito bite have been widely used to assess efficacy of preerythrocytic vaccine candidates in small proof-of-concept phase 2a clinical trials. Efficacy testing of blood-stage malaria parasite vaccines, however, has generally relied on larger-scale phase 2b field trials in malaria-endemic populations. We report the use of a blood-stage P. falciparum CHMI model to assess blood-stage vaccine candidates, using their impact on the parasite multiplication rate (PMR) as the primary efficacy end point. METHODS: Fifteen healthy United Kingdom adult volunteers were vaccinated with FMP2.1, a protein vaccine that is based on the 3D7 clone sequence of apical membrane antigen 1 (AMA1) and formulated in Adjuvant System 01 (AS01). Twelve vaccinees and 15 infectivity controls subsequently underwent blood-stage CHMI. Parasitemia was monitored by quantitative real-time polymerase chain reaction (PCR) analysis, and PMR was modeled from these data. RESULTS: FMP2.1/AS01 elicited anti-AMA1 T-cell and serum antibody responses. Analysis of purified immunoglobulin G showed functional growth inhibitory activity against P. falciparum in vitro. There were no vaccine- or CHMI-related safety concerns. All volunteers developed blood-stage parasitemia, with no impact of the vaccine on PMR. CONCLUSIONS: FMP2.1/AS01 demonstrated no efficacy after blood-stage CHMI. However, the model induced highly reproducible infection in all volunteers and will accelerate proof-of-concept testing of future blood-stage vaccine candidates. CLINICAL TRIALS REGISTRATION: NCT02044198.

A field trial to assess a blood-stage malaria vaccine.

BACKGROUND: Blood-stage malaria vaccines are intended to prevent clinical disease. The malaria vaccine FMP2.1/AS02(A), a recombinant protein based on apical membrane antigen 1 (AMA1) from the 3D7 strain of Plasmodium falciparum, has previously been shown to have immunogenicity and acceptable safety in Malian adults and children. METHODS: In a double-blind, randomized trial, we immunized 400 Malian children with either the malaria vaccine or a control (rabies) vaccine and followed them for 6 months. The primary end point was clinical malaria, defined as fever and at least 2500 parasites per cubic millimeter of blood. A secondary end point was clinical malaria caused by parasites with the AMA1 DNA sequence found in the vaccine strain. RESULTS: The cumulative incidence of the primary end point was 48.4% in the malaria-vaccine group and 54.4% in the control group; efficacy against the primary end point was 17.4% (hazard ratio for the primary end point, 0.83; 95% confidence interval [CI], 0.63 to 1.09; P=0.18). Efficacy against the first and subsequent episodes of clinical malaria, as defined on the basis of various parasite-density thresholds, was approximately 20%. Efficacy against clinical malaria caused by parasites with AMA1 corresponding to that of the vaccine strain was 64.3% (hazard ratio, 0.36; 95% CI, 0.08 to 0.86; P=0.03). Local reactions and fever after vaccination were more frequent with the malaria vaccine. CONCLUSIONS: On the basis of the primary end point, the malaria vaccine did not provide significant protection against clinical malaria, but on the basis of secondary results, it may have strain-specific efficacy. If this finding is confirmed, AMA1 might be useful in a multicomponent malaria vaccine. (Funded by the National Institute of Allergy and Infectious Diseases and others; ClinicalTrials.gov number, NCT00460525.).

Molecular basis of allele-specific efficacy of a blood-stage malaria vaccine: vaccine development implications.

The disappointing efficacy of blood-stage malaria vaccines may be explained in part by allele-specific immune responses that are directed against polymorphic epitopes on blood-stage antigens. FMP2.1/AS02(A), a blood-stage candidate vaccine based on apical membrane antigen 1 (AMA1) from the 3D7 strain of Plasmodium falciparum, had allele-specific efficacy against clinical malaria in a phase II trial in Malian children. We assessed the cross-protective efficacy of the malaria vaccine and inferred which polymorphic amino acid positions in AMA1 were the targets of protective allele-specific immune responses. FMP2.1/AS02(A) had the highest efficacy against AMA1 alleles that were identical to the 3D7 vaccine-type allele at 8 highly polymorphic amino acid positions in the cluster 1 loop (c1L) but differed from 3D7 elsewhere in the molecule. Comparison of the incidence of vaccine-type alleles before and after vaccination in the malaria vaccine and control groups and examination of the patterns of allele change at polymorphic positions in consecutive malaria episodes suggest that the highly polymorphic amino acid position 197 in c1L was the most critical determinant of allele-specific efficacy. These results indicate that a multivalent AMA1 vaccine with broad efficacy could include only a limited set of key alleles of this extremely polymorphic antigen.

Development of an improved blood-stage malaria vaccine targeting the essential RH5-CyRPA-RIPR invasion complex.

Reticulocyte-binding protein homologue 5 (RH5), a leading blood-stage Plasmodium falciparum malaria vaccine target, interacts with cysteine-rich protective antigen (CyRPA) and RH5-interacting protein (RIPR) to form an essential heterotrimeric "RCR-complex". We investigate whether RCR-complex vaccination can improve upon RH5 alone. Using monoclonal antibodies (mAbs) we show that parasite growth-inhibitory epitopes on each antigen are surface-exposed on the RCR-complex and that mAb pairs targeting different antigens can function additively or synergistically. However, immunisation of female rats with the RCR-complex fails to outperform RH5 alone due to immuno-dominance of RIPR coupled with inferior potency of anti-RIPR polyclonal IgG. We identify that all growth-inhibitory antibody epitopes of RIPR cluster within the C-terminal EGF-like domains and that a fusion of these domains to CyRPA, called "R78C", combined with RH5, improves the level of in vitro parasite growth inhibition compared to RH5 alone. These preclinical data justify the advancement of the RH5.1 + R78C/Matrix-M™ vaccine candidate to Phase 1 clinical trial.

Identification of minimal human MHC-restricted CD8+ T-cell epitopes within the Plasmodium falciparum circumsporozoite protein (CSP).

BACKGROUND: Plasmodium falciparum circumsporozoite protein (CSP) is a leading malaria vaccine candidate antigen, known to elicit protective antibody responses in humans (RTS,S vaccine). Recently, a DNA prime / adenovirus (Ad) vector boost vaccine encoding CSP and a second P. falciparum antigen, apical membrane antigen-1, also elicited sterile protection, but in this case associated with interferon gamma ELISpot and CD8+ T cell but not antibody responses. The finding that CSP delivered by an appropriate vaccine platform likely elicits protective cell-mediated immunity provided a rationale for identifying class I-restricted epitopes within this leading vaccine candidate antigen. METHODS: Limited samples of peripheral blood mononuclear cells from clinical trials of the Ad vaccine were used to identify CD8+ T cell epitopes within pools of overlapping 15mer peptides spanning portions of CSP that stimulated recall responses. Computerized algorithms (NetMHC) predicted 17 minimal class I-restricted 9-10mer epitopes within fifteen 15mers positive in ELISpot assay using PBMC from 10 HLA-matched study subjects. Four additional epitopes were subsequently predicted using NetMHC, matched to other study subjects without initial 15mer ELISpot screening. Nine of the putative epitopes were synthesized and tested by ELISpot assay, and six of these nine were further tested for CD8+ T cell responses by ELISpot CD4+ and CD8+ T cell-depletion and flow cytometry assays for evidence of CD8+ T cell dependence. RESULTS: Each of the nine putative epitopes, all sequence-conserved, recalled responses from HLA-matched CSP-immunized research subjects. Four shorter sequences contained within these sequences were identified using NetMHC predictions and may have contributed to recall responses. Five (9-10mer) epitopes were confirmed to be targets of CD8+ T cell responses using ELISpot depletion and ICS assays. Two 9mers among these nine epitopes were each restricted by two HLA supertypes (A01/B07; A01A24/A24) and one 9mer was restricted by three HLA supertypes (A01A24/A24/B27) indicating that some CSP class I-restricted epitopes, like DR epitopes, may be HLA-promiscuous. CONCLUSIONS: This study identified nine and confirmed five novel class I epitopes restricted by six HLA supertypes, suggesting that an adenovirus-vectored CSP vaccine would be immunogenic and potentially protective in genetically diverse populations.

Results from tandem Phase 1 studies evaluating the safety, reactogenicity and immunogenicity of the vaccine candidate antigen Plasmodium falciparum FVO merozoite surface protein-1 (MSP1(42)) administered intramuscularly with adjuvant system AS01.

BACKGROUND: The development of an asexual blood stage vaccine against Plasmodium falciparum malaria based on the major merozoite surface protein-1 (MSP1) antigen is founded on the protective efficacy observed in preclinical studies and induction of invasion and growth inhibitory antibody responses. The 42 kDa C-terminus of MSP1 has been developed as the recombinant protein vaccine antigen, and the 3D7 allotype, formulated with the Adjuvant System AS02A, has been evaluated extensively in human clinical trials. In preclinical rabbit studies, the FVO allele of MSP142 has been shown to have improved immunogenicity over the 3D7 allele, in terms of antibody titres as well as growth inhibitory activity of antibodies against both the heterologous 3D7 and homologous FVO parasites. METHODS: Two Phase 1 clinical studies were conducted to examine the safety, reactogenicity and immunogenicity of the FVO allele of MSP142 in the adjuvant system AS01 administered intramuscularly at 0-, 1-, and 2-months: one in the USA and, after evaluation of safety data results, one in Western Kenya. The US study was an open-label, dose escalation study of 10 and 50 µg doses of MSP142 in 26 adults, while the Kenya study, evaluating 30 volunteers, was a double-blind, randomized study of only the 50 µg dose with a rabies vaccine comparator. RESULTS: In these studies it was demonstrated that this vaccine formulation has an acceptable safety profile and is immunogenic in malaria-naïve and malaria-experienced populations. High titres of anti-MSP1 antibodies were induced in both study populations, although there was a limited number of volunteers whose serum demonstrated significant inhibition of blood-stage parasites as measured by growth inhibition assay. In the US volunteers, the antibodies generated exhibited better cross-reactivity to heterologous MSP1 alleles than a MSP1-based vaccine (3D7 allele) previously tested at both study sites. CONCLUSIONS: Given that the primary effector mechanism for blood stage vaccine targets is humoral, the antibody responses demonstrated to this vaccine candidate, both quantitative (total antibody titres) and qualitative (functional antibodies inhibiting parasite growth) warrant further consideration of its application in endemic settings. TRIAL REGISTRATIONS: Clinical Trials NCT00666380.

Restricted valency (NPNA)n repeats and junctional epitope-based circumsporozoite protein vaccines against Plasmodium falciparum.

The Circumsporozoite Protein (CSP) of Plasmodium falciparum contains an N-terminal region, a conserved Region I (RI), a junctional region, 25-42 copies of major (NPNA) and minor repeats followed by a C-terminal domain. The recently approved malaria vaccine, RTS,S/AS01 contains NPNAx19 and the C-terminal region of CSP. The efficacy of RTS,S against natural infection is low and short-lived, and mapping epitopes of inhibitory monoclonal antibodies may allow for rational improvement of CSP vaccines. Tobacco Mosaic Virus (TMV) was used here to display the junctional epitope (mAb CIS43), Region I (mAb 5D5), NPNAx5, and NPNAx20 epitope of CSP (mAbs 317 and 580). Protection studies in mice revealed that Region I did not elicit protective antibodies, and polyclonal antibodies against the junctional epitope showed equivalent protection to NPNAx5. Combining the junctional and NPNAx5 epitopes reduced immunogenicity and efficacy, and increasing the repeat valency to NPNAx20 did not improve upon NPNAx5. TMV was confirmed as a versatile vaccine platform for displaying small epitopes defined by neutralizing mAbs. We show that polyclonal antibodies against engineered VLPs can recapitulate the binding specificity of the mAbs and immune-focusing by reducing the structural complexity of an epitope may be superior to immune-broadening as a vaccine design approach. Most importantly the junctional and restricted valency NPNA epitopes can be the basis for developing highly effective second-generation malaria vaccine candidates.

First-in-human assessment of safety and immunogenicity of low and high doses of Plasmodium falciparum malaria protein 013 (FMP013) administered intramuscularly with ALFQ adjuvant in healthy malaria-naïve adults.

The global burden of malaria remains substantial. Circumsporozoite protein (CSP) has been demonstrated to be an effective target antigen, however, improvements that offer more efficacious and more durable protection are still needed. In support of research and development of next-generation malaria vaccines, Walter Reed Army Institute of Research (WRAIR) has developed a CSP-based antigen (FMP013) and a novel adjuvant ALFQ (Army Liposome Formulation containing QS-21). We present a single center, open-label, dose-escalation Phase 1 clinical trial to evaluate the safety and immunogenicity of the FMP013/ALFQ malaria vaccine candidate. In this first-in-human evaluation of both the antigen and adjuvant, we enrolled ten subjects; five received 20 µg FMP013 / 0.5 mL ALFQ (Low dose group), and five received 40 µg FMP013 / 1.0 mL ALFQ (High dose group) on study days 1, 29, and 57. Adverse events and immune responses were assessed during the study period. The clinical safety profile was acceptable and there were no serious adverse events. Both groups exhibited robust humoral and cellular immunological responses, and compared favorably with historical responses reported for RTS,S/AS01. Based on a lower reactogenicity profile, the 20 µg FMP013 / 0.5 mL ALFQ (Low dose) was selected for follow-on efficacy testing by controlled human malaria infection (CHMI) with a separate cohort. Trial Registration:Clinicaltrials.gov Identifier NCT04268420 (Registered February 13, 2020).

RH5.1-CyRPA-Ripr antigen combination vaccine shows little improvement over RH5.1 in a preclinical setting.

Background: RH5 is the leading vaccine candidate for the Plasmodium falciparum blood stage and has shown impact on parasite growth in the blood in a human clinical trial. RH5 binds to Ripr and CyRPA at the apical end of the invasive merozoite form, and this complex, designated RCR, is essential for entry into human erythrocytes. RH5 has advanced to human clinical trials, and the impact on parasite growth in the blood was encouraging but modest. This study assessed the potential of a protein-in-adjuvant blood stage malaria vaccine based on a combination of RH5, Ripr and CyRPA to provide improved neutralizing activity against P. falciparum in vitro. Methods: Mice were immunized with the individual RCR antigens to down select the best performing adjuvant formulation and rats were immunized with the individual RCR antigens to select the correct antigen dose. A second cohort of rats were immunized with single, double and triple antigen combinations to assess immunogenicity and parasite neutralizing activity in growth inhibition assays. Results: The DPX® platform was identified as the best performing formulation in potentiating P. falciparum inhibitory antibody responses to these antigens. The three antigens derived from RH5, Ripr and CyRPA proteins formulated with DPX induced highly inhibitory parasite neutralising antibodies. Notably, RH5 either as a single antigen or in combination with Ripr and/or CyRPA, induced inhibitory antibodies that outperformed CyRPA, Ripr. Conclusion: An RCR combination vaccine may not induce substantially improved protective immunity as compared with RH5 as a single immunogen in a clinical setting and leaves the development pathway open for other antigens to be combined with RH5 as a next generation malaria vaccine.

Reduced blood-stage malaria growth and immune correlates in humans following RH5 vaccination.

BACKGROUND: Development of an effective vaccine against the pathogenic blood-stage infection of human malaria has proved challenging, and no candidate vaccine has affected blood-stage parasitemia following controlled human malaria infection (CHMI) with blood-stage Plasmodium falciparum. METHODS: We undertook a phase I/IIa clinical trial in healthy adults in the United Kingdom of the RH5.1 recombinant protein vaccine, targeting the P. falciparum reticulocyte-binding protein homolog 5 (RH5), formulated in AS01B adjuvant. We assessed safety, immunogenicity, and efficacy against blood-stage CHMI. Trial registered at ClinicalTrials.gov, NCT02927145. FINDINGS: The RH5.1/AS01B formulation was administered using a range of RH5.1 protein vaccine doses (2, 10, and 50 µg) and was found to be safe and well tolerated. A regimen using a delayed and fractional third dose, in contrast to three doses given at monthly intervals, led to significantly improved antibody response longevity over ∼2 years of follow-up. Following primary and secondary CHMI of vaccinees with blood-stage P. falciparum, a significant reduction in parasite growth rate was observed, defining a milestone for the blood-stage malaria vaccine field. We show that growth inhibition activity measured in vitro using purified immunoglobulin G (IgG) antibody strongly correlates with in vivo reduction of the parasite growth rate and also identify other antibody feature sets by systems serology, including the plasma anti-RH5 IgA1 response, that are associated with challenge outcome. CONCLUSIONS: Our data provide a new framework to guide rational design and delivery of next-generation vaccines to protect against malaria disease. FUNDING: This study was supported by USAID, UK MRC, Wellcome Trust, NIAID, and the NIHR Oxford-BRC.

A three-antigen Plasmodium falciparum DNA prime-Adenovirus boost malaria vaccine regimen is superior to a two-antigen regimen and protects against controlled human malaria infection in healthy malaria-naïve adults.

BACKGROUND: A DNA-prime/human adenovirus serotype 5 (HuAd5) boost vaccine encoding Plasmodium falciparum (Pf) circumsporozoite protein (PfCSP) and Pf apical membrane antigen-1 (PfAMA1), elicited protection in 4/15 (27%) of subjects against controlled human malaria infection (CHMI) that was statistically associated with CD8+ T cell responses. Subjects with high level pre-existing immunity to HuAd5 were not protected, suggesting an adverse effect on vaccine efficacy (VE). We replaced HuAd5 with chimpanzee adenovirus 63 (ChAd63), and repeated the study, assessing both the two-antigen (CSP, AMA1 = CA) vaccine, and a novel three-antigen (CSP, AMA1, ME-TRAP = CAT) vaccine that included a third pre-erythrocytic stage antigen [malaria multiple epitopes (ME) fused to the Pf thrombospondin-related adhesive protein (TRAP)] to potentially enhance protection. METHODOLOGY: This was an open label, randomized Phase 1 trial, assessing safety, tolerability, and VE against CHMI in healthy, malaria naïve adults. Forty subjects (20 each group) were to receive three monthly CA or CAT DNA priming immunizations, followed by corresponding ChAd63 boost four months later. Four weeks after the boost, immunized subjects and 12 infectivity controls underwent CHMI by mosquito bite using the Pf3D7 strain. VE was assessed by determining the differences in time to parasitemia as detected by thick blood smears up to 28-days post CHMI and utilizing the log rank test, and by calculating the risk ratio of each treatment group and subtracting from 1, with significance calculated by the Cochran-Mantel-Haenszel method. RESULTS: In both groups, systemic adverse events (AEs) were significantly higher after the ChAd63 boost than DNA immunizations. Eleven of 12 infectivity controls developed parasitemia (mean 11.7 days). In the CA group, 15 of 16 (93.8%) immunized subjects developed parasitemia (mean 12.0 days). In the CAT group, 11 of 16 (63.8%) immunized subjects developed parasitemia (mean 13.0 days), indicating significant protection by log rank test compared to infectivity controls (p = 0.0406) and the CA group (p = 0.0229). VE (1 minus the risk ratio) in the CAT group was 25% compared to -2% in the CA group. The CA and CAT vaccines induced robust humoral (ELISA antibodies against CSP, AMA1 and TRAP, and IFA responses against sporozoites and Pf3D7 blood stages), and cellular responses (IFN-γ FluoroSpot responses to CSP, AMA1 and TRAP) that were not associated with protection. CONCLUSIONS: This study demonstrated that the ChAd63 CAT vaccine exhibited significant protective efficacy, and confirmed protection was afforded by adding a third antigen (T) to a two-antigen (CA) formulation to achieve increased VE. Although the ChAd63-CAT vaccine was associated with increased frequencies of systemic AEs compared to the CA vaccine and, historically, compared to the HuAd5 vectored malaria vaccine encoding CSP and AMA1, they were transient and associated with increased vector dosing.

A malaria vaccine protects Aotus monkeys against virulent Plasmodium falciparum infection.

The Plasmodium falciparum protein, apical membrane antigen 1 forms a complex with another parasite protein, rhoptry neck protein 2, to initiate junction formation with the erythrocyte and is essential for merozoite invasion during the blood stage of infection. Consequently, apical membrane antigen 1 has been a target of vaccine development but vaccination with apical membrane antigen 1 alone in controlled human malaria infections failed to protect and showed limited efficacy in field trials. Here we show that vaccination with AMA1-RON2L complex in Freund's adjuvant protects Aotus monkeys against a virulent Plasmodium falciparum infection. Vaccination with AMA1 alone gave only partial protection, delaying infection in one of eight animals. However, the AMA1-RON2L complex vaccine completely protected four of eight monkeys and substantially delayed infection (>25 days) in three of the other four animals. Interestingly, antibodies from monkeys vaccinated with the AMA1-RON2L complex had significantly higher neutralizing activity than antibodies from monkeys vaccinated with AMA1 alone. Importantly, we show that antibodies from animals vaccinated with the complex have significantly higher neutralization activity against non-vaccine type parasites. We suggest that vaccination with the AMA1-RON2L complex induces functional antibodies that better recognize AMA1 as it appears complexed with RON2 during merozoite invasion. These data justify progression of this next generation AMA1 vaccine towards human trials.

Strain-specific Plasmodium falciparum growth inhibition among Malian children immunized with a blood-stage malaria vaccine.

The blood-stage malaria vaccine FMP2.1/AS02A, comprised of recombinant Plasmodium falciparum apical membrane antigen 1 (AMA1) and the adjuvant system AS02A, had strain-specific efficacy against clinical malaria caused by P. falciparum with the vaccine strain 3D7 AMA1 sequence. To evaluate a potential correlate of protection, we measured the ability of participant sera to inhibit growth of 3D7 and FVO strains in vitro using high-throughput growth inhibition assay (GIA) testing. Sera from 400 children randomized to receive either malaria vaccine or a control rabies vaccine were assessed at baseline and over two annual malaria transmission seasons after immunization. Baseline GIA against vaccine strain 3D7 and FVO strain was similar in both groups, but more children in the malaria vaccine group than in the control group had 3D7 and FVO GIA activity ≥15% 30 days after the last vaccination (day 90) (49% vs. 16%, p<0.0001; and 71.8% vs. 60.4%, p = 0.02). From baseline to day 90, 3D7 GIA in the vaccine group was 7.4 times the mean increase in the control group (p<0.0001). In AMA1 vaccinees, 3D7 GIA activity subsequently returned to baseline one year after vaccination (day 364) and did not correlate with efficacy in the extended efficacy time period to day 730. In Cox proportional hazards regression models with time-varying covariates, there was a slight suggestion of an association between 3D7 GIA activity and increased risk of clinical malaria between day 90 and day 240. We conclude that vaccination with this AMA1-based malaria vaccine increased inhibition of parasite growth, but this increase was not associated with allele-specific efficacy in the first malaria season. These results provide a framework for testing functional immune correlates of protection against clinical malaria in field trials, and will help to guide similar analyses for next-generation malaria vaccines. Clinical trials registry: This clinical trial was registered on clinicaltrials.gov, registry number NCT00460525.

Vaccine Strain-Specificity of Protective HLA-Restricted Class 1 P. falciparum Epitopes.

A DNA prime/adenovirus boost malaria vaccine encoding Plasmodium falciparum strain 3D7 CSP and AMA1 elicited sterile clinical protection associated with CD8+ T cell interferon-gamma (IFN-γ) cells responses directed to HLA class 1-restricted AMA1 epitopes of the vaccine strain 3D7. Since a highly effective malaria vaccine must be broadly protective against multiple P. falciparum strains, we compared these AMA1 epitopes of two P. falciparum strains (7G8 and 3D7), which differ by single amino acid substitutions, in their ability to recall CD8+ T cell activities using ELISpot and flow cytometry/intracellular staining assays. The 7G8 variant peptides did not recall 3D7 vaccine-induced CD8+ T IFN-γ cell responses in these assays, suggesting that protection may be limited to the vaccine strain. The predicted MHC binding affinities of the 7G8 variant epitopes were similar to the 3D7 epitopes, suggesting that the amino acid substitutions of the 7G8 variants may have interfered with TCR recognition of the MHC:peptide complex or that the 7G8 variant may have acted as an altered peptide ligand. These results stress the importance of functional assays in defining protective epitopes. Clinical Trials Registrations: NCT00870987, NCT00392015.

Correction: Vaccine Strain-Specificity of Protective HLA-Restricted Class 1 P. falciparum Epitopes.

[This corrects the article DOI: 10.1371/journal.pone.0163026.].

Strain-specific Plasmodium falciparum multifunctional CD4(+) T cell cytokine expression in Malian children immunized with the FMP2.1/AS02A vaccine candidate.

Based on Plasmodium falciparum (Pf) apical membrane antigen 1 (AMA1) from strain 3D7, the malaria vaccine candidate FMP2.1/AS02A showed strain-specific efficacy in a Phase 2 clinical trial in 400 Malian children randomized to 3 doses of the AMA1 vaccine candidate or control rabies vaccine on days 0, 30 and 60. A subset of 10 Pf(-) (i.e., no clinical malaria episodes) AMA1 recipients, 11 Pf(+) (clinical malaria episodes with parasites with 3D7 or Fab9-type AMA1 cluster 1 loop [c1L]) AMA1 recipients, and 10 controls were randomly chosen for analysis. Peripheral blood mononuclear cells (PBMCs) isolated on days 0, 90 and 150 were stimulated with full-length 3D7 AMA1 and c1L from strains 3D7 (c3D7) and Fab9 (cFab9). Production of IFN-γ, TNF-α, IL-2, and/or IL-17A was analyzed by flow cytometry. Among AMA1 recipients, 18/21 evaluable samples stimulated with AMA1 demonstrated increased IFN-γ, TNF-α, and IL-2 derived from CD4(+) T cells by day 150 compared to 0/10 in the control group (p<0.0001). Among AMA1 vaccines, CD4(+) cells expressing both TNF-α and IL-2 were increased in Pf(-) children compared to Pf(+) children. When PBMCs were stimulated with c3D7 and cFab9 separately, 4/18 AMA1 recipients with an AMA1-specific CD4(+) response had a significant response to one or both c1L. This suggests that recognition of the AMA1 antigen is not dependent upon c1L alone. In summary, AMA1-specific T cell responses were notably increased in children immunized with an AMA1-based vaccine candidate. The role of CD4(+)TNF-α(+)IL-2(+)-expressing T cells in vaccine-induced strain-specific protection against clinical malaria requires further exploration. Clinicaltrials.gov Identifier: NCT00460525.

A multilateral effort to develop DNA vaccines against falciparum malaria.

Scientists from several organizations worldwide are working together to develop a multistage, multigene DNA-based vaccine against Plasmodium falciparum malaria. This collaborative vaccine development effort is named Multi-Stage DNA-based Malaria Vaccine Operation. An advisory board of international experts in vaccinology, malariology and field trials provides the scientific oversight to support the operation. This article discusses the rationale for the approach, underlying concepts and the pre-clinical development process, and provides a brief outline of the plans for the clinical testing of a multistage, multiantigen malaria vaccine based on DNA plasmid immunization technology.

Sterile immunity to malaria after DNA prime/adenovirus boost immunization is associated with effector memory CD8+T cells targeting AMA1 class I epitopes.

BACKGROUND: Fifteen volunteers were immunized with three doses of plasmid DNA encoding P. falciparum circumsporozoite protein (CSP) and apical membrane antigen-1 (AMA1) and boosted with human adenovirus-5 (Ad) expressing the same antigens (DNA/Ad). Four volunteers (27%) demonstrated sterile immunity to controlled human malaria infection and, overall, protection was statistically significantly associated with ELISpot and CD8+ T cell IFN-γ activities to AMA1 but not CSP. DNA priming was required for protection, as 18 additional subjects immunized with Ad alone (AdCA) did not develop sterile protection. METHODOLOGY/PRINCIPAL FINDINGS: We sought to identify correlates of protection, recognizing that DNA-priming may induce different responses than AdCA alone. Among protected volunteers, two and three had higher ELISpot and CD8+ T cell IFN-γ responses to CSP and AMA1, respectively, than non-protected volunteers. Unexpectedly, non-protected volunteers in the AdCA trial showed ELISpot and CD8+ T cell IFN-γ responses to AMA1 equal to or higher than the protected volunteers. T cell functionality assessed by intracellular cytokine staining for IFN-γ, TNF-α and IL-2 likewise did not distinguish protected from non-protected volunteers across both trials. However, three of the four protected volunteers showed higher effector to central memory CD8+ T cell ratios to AMA1, and one of these to CSP, than non-protected volunteers for both antigens. These responses were focused on discrete regions of CSP and AMA1. Class I epitopes restricted by A*03 or B*58 supertypes within these regions of AMA1 strongly recalled responses in three of four protected volunteers. We hypothesize that vaccine-induced effector memory CD8+ T cells recognizing a single class I epitope can confer sterile immunity to P. falciparum in humans. CONCLUSIONS/SIGNIFICANCE: We suggest that better understanding of which epitopes within malaria antigens can confer sterile immunity and design of vaccine approaches that elicit responses to these epitopes will increase the potency of next generation gene-based vaccines.