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Abstract Background To prepare field sites for malaria vaccine trials, it is important to determine baseline antibody and T cell responses to candidate malaria vaccine antigens. Assessing T cell responses is especially challenging, given genetic restriction, low responses observed in endemic areas, their variability over time, potential suppression by parasitaemia and the intrinsic variability of the assays. Methods In Part A of this study, antibody titres were measured in adults from urban and rural communities in Ghana to recombinant Plasmodium falciparum CSP, SSP2/TRAP, LSA1, EXP1, MSP1, MSP3 and EBA175 by ELISA, and to sporozoites and infected erythrocytes by IFA. Positive ELISA responses were determined using two methods. T cell responses to defined CD8 or CD4 T cell epitopes from CSP, SSP2/TRAP, LSA1 and EXP1 were measured by ex vivo IFN-γ ELISpot assays using HLA-matched Class I- and DR-restricted synthetic peptides. In Part B, the reproducibility of the ELISpot assay to CSP and AMA1 was measured by repeating assays of individual samples using peptide pools and low, medium or high stringency criteria for defining positive responses, and by comparing samples collected two weeks apart. Results In Part A, positive antibody responses varied widely from 17%-100%, according to the antigen and statistical method, with blood stage antigens showing more frequent and higher magnitude responses. ELISA titres were higher in rural subjects, while IFA titres and the frequencies and magnitudes of ex vivo ELISpot activities were similar in both communities. DR-restricted peptides showed stronger responses than Class I-restricted peptides. In Part B, the most stringent statistical criteria gave the fewest, and the least stringent the most positive responses, with reproducibility slightly higher using the least stringent method when assays were repeated. Results varied significantly between the two-week time-points for many participants. Conclusions All participants were positive for at least one malaria protein by ELISA, with results dependent on the criteria for positivity. Likewise, ELISpot responses varied among participants, but were relatively reproducible by the three methods tested, especially the least stringent, when assays were repeated. However, results often differed between samples taken two weeks apart, indicating significant biological variability over short intervals.

Abstract Background Despite years of effort, a licensed malaria vaccine is not yet available. One of the obstacles facing the development of a malaria vaccine is the extensive heterogeneity of many of the current malaria vaccine antigens. To counteract this antigenic diversity, an effective malaria vaccine may need to elicit an immune response against multiple malaria antigens, thereby limiting the negative impact of variability in any one antigen. Since most of the malaria vaccine antigens that have been evaluated in people have not elicited a protective immune response, there is a need to identify additional protective antigens. In this study, the efficacy of three pre-erythrocytic stage malaria antigens was evaluated in a Plasmodium yoelii/mouse protection model. Methods Mice were immunized with plasmid DNA and vaccinia virus vectors that expressed one, two or all three P. yoelii vaccine antigens. The immunized mice were challenged with 300 P. yoelii sporozoites and evaluated for subsequent infection. Results Vaccines that expressed any one of the three antigens did not protect a high percentage of mice against a P. yoelii challenge. However, vaccines that expressed all three antigens protected a higher percentage of mice than a vaccine that expressed PyCSP, the most efficacious malaria vaccine antigen. Dissection of the multi-antigen vaccine indicated that protection was primarily associated with two of the three P. yoelii antigens. The protection elicited by a vaccine expressing these two antigens exceeded the sum of the protection elicited by the single antigen vaccines, suggesting a potential synergistic interaction. Conclusions This work identifies two promising malaria vaccine antigen candidates and suggests that a multi-antigen vaccine may be more efficacious than a single antigen vaccine.

Abstract Background Plasmodium falciparum apical membrane antigen-1 (AMA1) is a leading malaria vaccine candidate antigen that is expressed by sporozoite, liver and blood stage parasites. Since CD8+ T cell responses have been implicated in protection against pre-erythrocytic stage malaria, this study was designed to identify MHC class I-restricted epitopes within AMA1. Methods A recombinant adenovirus serotype 5 vector expressing P. falciparum AMA1 was highly immunogenic when administered to healthy, malaria-naive adult volunteers as determined by IFN-γ ELISpot responses to peptide pools containing overlapping 15-mer peptides spanning full-length AMA1. Computerized algorithms (NetMHC software) were used to predict minimal MHC-restricted 8-10-mer epitope sequences within AMA1 15-mer peptides active in ELISpot. A subset of epitopes was synthesized and tested for induction of CD8+ T cell IFN-γ responses by ELISpot depletion and ICS assays. A 3-dimensional model combining Domains I + II of P. falciparum AMA1 and Domain III of P. vivax AMA1 was used to map these epitopes. Results Fourteen 8-10-mer epitopes were predicted to bind to HLA supertypes A01 (3 epitopes), A02 (4 epitopes), B08 (2 epitopes) and B44 (5 epitopes). Nine of the 14 predicted epitopes were recognized in ELISpot or ELISpot and ICS assays by one or more volunteers. Depletion of T cell subsets confirmed that these epitopes were CD8+ T cell-dependent. A mixture of the 14 minimal epitopes was capable of recalling CD8+ T cell IFN-γ responses from PBMC of immunized volunteers. Thirteen of the 14 predicted epitopes were polymorphic and the majority localized to the more conserved front surface of the AMA1 model structure. Conclusions This study predicted 14 and confirmed nine MHC class I-restricted CD8+ T cell epitopes on AMA1 recognized in the context of seven HLA alleles. These HLA alleles belong to four HLA supertypes that have a phenotypic frequency between 23% - 100% in different human populations.

Background: Malaria eradication requires a combined effort involving all available control tools, and these efforts would be complemented by an effective vaccine. The antigen targets of immune responses may show polymorphisms that can undermine their recognition by immune effectors and hence render vaccines based on antigens from a single parasite variant ineffective against other variants. This study compared the influence of allelic polymorphisms in Plasmodium falciparum apical membrane antigen 1 (PfAMA1) peptide sequences from three strains of P. falciparum (3D7, 7G8 and FVO) on their function as immunodominant targets of T cell responses in high and low malaria transmission communities in Ghana., Methods: Peripheral blood mononuclear cells (PBMCs) from 10 subjects from a high transmission area (Obom) and 10 subjects from a low transmission area (Legon) were tested against 15 predicted CD8 + T cell minimal epitopes within the PfAMA1 antigen of multiple parasite strains using IFN-γ ELISpot assay. The peptides were also tested in similar assays against CD8 + enriched PBMC fractions from the same subjects in an effort to characterize the responding T cell subsets., Results: In assays using unfractionated PBMCs, two subjects from the high transmission area, Obom, responded positively to four (26.7%) of the 15 tested peptides. None of the Legon subject PBMCs yielded positive peptide responses using unfractionated PBMCs. In assays with CD8 + enriched PBMCs, three subjects from Obom made positive recall responses to six (40%) of the 15 tested peptides, while only one subject from Legon made a positive recall response to a single peptide. Overall, 5 of the 20 study subjects who had positive peptide-specific IFN-γ recall responses were from the high transmission area, Obom. Furthermore, while subjects from Obom responded to peptides in PfAMA1 from multiple parasite strains, one subject from Legon responded to a peptide from 3D7 strain only., Conclusions: The current data demonstrate the possibility of a real effect of PfAMA1 polymorphisms on the induction of T cell responses in malaria exposed subjects, and this effect may be more pronounced in communities with higher parasite exposure., (© 2021. The Author(s).)

Background: Malaria remains the top infectious disease threat facing the U.S. military in many forward operating environments. Compliance with malaria chemoprophylaxis remains a critical component in preventing malaria in the deployed Service Member. Studies of previous military operations show that compliance is consistently higher with weekly versus daily dosing regimens. Current FDA approved weekly chemoprophylaxis options have contraindications that can limit prescribing. The combination of chloroquine (CQ) with azithromycin (AZ) has previously been shown to be an efficacious treatment option for malaria, has pharmacokinetics compatible with weekly dosing, and has shown synergy when combined in vitro., Methods: In this open label study, 18 healthy volunteers, aged 18-50 years (inclusive), were randomly assigned to receive either 300 mg CQ or 300 mg CQ and 2 gm azithromycin (CQAZ) of directly observed therapy, weekly for 3 weeks prior to undergoing mosquito bite challenge with chloroquine-resistant Plasmodium falciparum. Volunteers that remained asymptomatic and had no evidence of parasitaemia continued to receive weekly post-exposure chemoprophylaxis for 3 weeks following malaria challenge. The primary endpoint was the number of volunteers that remained asymptomatic and had no evidence of parasitaemia 28 days after the malaria challenge., Results: All 6 (100%) volunteers randomized to the CQ control group became symptomatic with parasitaemia during the 28-day post-challenge period. Only 1/12 (8.3%) of volunteers in the CQAZ group developed symptoms and parasitaemia during the 28-day post-challenge period. However, after chemoprophylaxis was discontinued an additional 6 volunteers developed parasitaemia between days 28-41 after challenge, with 4 of 6 experiencing symptoms. 80% of subjects in the CQAZ group experienced treatment related gastrointestinal adverse events (including 13% that experienced severe nausea) compared to 38% in the CQ group. A comparison of the pharmacokinetics in the CQAZ group demonstrated higher azithromycin Cmax (p = 0.03) and AUC (p = 0.044) levels in those volunteers who never became parasitaemic compared to those who did., Conclusion: Given the high rate of side effects and poor efficacy when administered for 3 weeks before and after challenge, the combination of weekly chloroquine and azithromycin is a suboptimal regimen combination for weekly malaria chemoprophylaxis. Trial registration ClinicalTrials.gov NCT03278808.

Background: A DNA-human Ad5 (HuAd5) prime-boost malaria vaccine has been shown to protect volunteers against a controlled human malaria infection. The potency of this vaccine, however, appeared to be affected by the presence of pre-existing immunity against the HuAd5 vector. Since HuAd5 seroprevalence is very high in malaria-endemic areas of the world, HuAd5 may not be the most appropriate malaria vaccine vector. This report describes the evaluation of the seroprevalence, immunogenicity and efficacy of three newly identified gorilla adenoviruses, GC44, GC45 and GC46, as potential malaria vaccine vectors., Results: The seroprevalence of GC44, GC45 and GC46 is very low, and the three vectors are not efficiently neutralized by human sera from Kenya and Ghana, two countries where malaria is endemic. In mice, a single administration of GC44, GC45 and GC46 vectors expressing a murine malaria gene, Plasmodium yoelii circumsporozoite protein (PyCSP), induced robust PyCSP-specific T cell and antibody responses that were at least as high as a comparable HuAd5-PyCSP vector. Efficacy studies in a murine malaria model indicated that a prime-boost regimen with DNA-PyCSP and GC-PyCSP vectors can protect mice against a malaria challenge. Moreover, these studies indicated that a DNA-GC46-PyCSP vaccine regimen was significantly more efficacious than a DNA-HuAd5-PyCSP regimen., Conclusion: These data suggest that these gorilla-based adenovectors have key performance characteristics for an effective malaria vaccine. The superior performance of GC46 over HuAd5 highlights its potential for clinical development.

Background: In this phase 1 clinical trial, healthy adult, malaria-naïve subjects were immunized with radiation-attenuated Plasmodium falciparum sporozoites (PfRAS) by mosquito bite and then underwent controlled human malaria infection (CHMI). The PfRAS model for immunization against malaria had previously induced >90 % sterile protection against homologous CHMI. This study was to further explore the safety, tolerability and protective efficacy of the PfRAS model and to provide biological specimens to characterize protective immune responses and identify protective antigens in support of malaria vaccine development., Methods: Fifty-seven subjects were screened, 41 enrolled and 30 received at least one immunization. The true-immunized subjects received PfRAS via mosquito bite and the mock-immunized subjects received mosquito bites from irradiated uninfected mosquitoes. Sera and peripheral blood mononuclear cells (PBMCs) were collected before and after PfRAS immunizations., Results: Immunization with PfRAS was generally safe and well tolerated, and repeated immunization via mosquito bite did not appear to increase the risk or severity of AEs. Local adverse events (AEs) of true-immunized and mock-immunized groups consisted of erythaema, papules, swelling, and induration and were consistent with reactions from mosquito bites seen in nature. Two subjects, one true- and one mock-immunized, developed large local reactions that completely resolved, were likely a result of mosquito salivary antigens, and were withdrawn from further participation as a safety precaution. Systemic AEs were generally rare and mild, consisting of headache, myalgia, nausea, and low-grade fevers. Two true-immunized subjects experienced fever, malaise, myalgia, nausea, and rigours approximately 16 h after immunization. These symptoms likely resulted from pre-formed antibodies interacting with mosquito salivary antigens. Ten subjects immunized with PfRAS underwent CHMI and five subjects (50 %) were sterilely protected and there was a significant delay to parasitaemia in the other five subjects. All ten subjects developed humoral immune responses to whole sporozoites and to the circumsporozoite protein prior to CHMI, although the differences between protected and non-protected subjects were not statistically significant for this small sample size., Conclusions: The protective efficacy of this clinical trial (50 %) was notably less than previously reported (>90 %). This may be related to differences in host genetics or the inherent variability in mosquito biting behavior and numbers of sporozoites injected. Differences in trial procedures, such as the use of leukapheresis prior to CHMI and of a longer interval between the final immunization and CHMI in these subjects compared to earlier trials, may also have reduced protective efficacy. This trial has been retrospectively registered at ISRCTN ID 17372582, May 31, 2016.

Background: Malaria eradication requires a concerted approach involving all available control tools, and an effective vaccine would complement these efforts. An effective malaria vaccine should be able to induce protective immune responses in a genetically diverse population. Identification of immunodominant T cell epitopes will assist in determining if candidate vaccines will be immunogenic in malaria-endemic areas. This study therefore investigated whether class I-restricted T cell epitopes of two leading malaria vaccine antigens, Plasmodium falciparum circumsporozoite protein (CSP) and apical membrane antigen-1 (AMA1), could recall T cell interferon-γ responses from naturally exposed subjects using ex vivo ELISpot assays., Methods: Thirty-five subjects aged between 24 and 43 years were recruited from a malaria-endemic urban community of Ghana in 2011, and their peripheral blood mononuclear cells (PBMCs) were tested in ELISpot IFN-γ assays against overlapping 15mer peptide pools spanning the entire CSP and AMA1 antigens, and 9-10mer peptide epitope mixtures that included previously identified and/or predicted human leukocyte antigen (HLA) class 1-restricted epitopes from same two antigens., Results: For CSP, 26 % of subjects responded to at least one of the nine 15mer peptide pools whilst 17 % responded to at least one of the five 9-10mer HLA-restricted epitope mixtures. For AMA1, 63 % of subjects responded to at least one of the 12 AMA1 15mer peptide pools and 51 % responded to at least one of the six 9-10mer HLA-restricted epitope mixtures. Following analysis of data from the two sets of peptide pools, along with bioinformatics predictions of class I-restricted epitopes and the HLA supertypes expressed by a subset of study subjects, peptide pools that may contain epitopes recognized by multiple HLA supertypes were identified. Collectively, these results suggest that natural transmission elicits ELISpot IFN-γ activities to class 1-restricted epitopes that are largely HLA-promiscuous., Conclusions: These results generally demonstrate that CSP and AMA1 peptides recalled ELISpot IFN-γ responses from naturally exposed individuals and that both CSP and AMA1 contain diverse class 1-restricted epitopes that are HLA-promiscuous and are widely recognized in this population.

Background: A malaria vaccine that targets the sporozoite/liver stage parasites could potentially prevent blood stage infection and the associated clinical symptoms. Identification of sporozoite/liver stage antigens is, therefore, crucial for the development of effective vaccines. Cell-traversal protein for ookinetes and sporozoites (CelTOS) is a highly conserved antigen involved in sporozoite motility and hepatocyte invasion and has been shown to induce significant IFN-γ production in PBMCs from radiation-attenuated sporozoite-immunized malaria-naïve individuals. The aim of this study was to ascertain whether such CelTOS-specific recall responses are also induced in individuals with natural exposure to Plasmodium falciparum., Methods: Ex vivo IFN-γ responses to 15mer overlapping peptide pools covering the entire sequence of CelTOS and five other candidate antigens, CSP, AMA1, MSP1, TRAP and LSA1, were characterized using PBMCs from 35 malaria exposed adults. Responses to four CelTOS peptide pools (CelTp1, CelTp2, CelTp3 and CelTp4), a pool containing peptides from the entire CelTOS antigen (CelTTp), and pools comprised of overlapping peptides from each of the other five malaria antigens were assessed by ex vivo ELISpot assay. A positive IFN-γ response for stimulants was defined by two criteria; a stimulation index of two or greater relative to the unstimulated control, and a difference of 10 or greater in spot forming cells between stimulant and the unstimulated control., Results: Of the 35 volunteers tested, five had positive IFN-γ recall responses against the four different CelTOS pools while four volunteers made responses against the CelTTp pool; six volunteers were, therefore, positive with CelTOS. By contrast, six volunteers responded to AMA1, seven to LSA1, 15 to MSP1 and two volunteers responded against CSP and TRAP., Conclusions: These results suggest natural malaria transmission induces CelTOS-specific ex vivo IFN-γ in Ghanaian adults and that the frequency of these responses was similar to those of other previously characterized malaria antigens. These findings support the further evaluation of CelTOS as a pre-erythrocytic candidate antigen for inclusion in a potential multi-antigen vaccine.

Background: Reported malaria cases continue to decline globally, and this has been attributed to strategic implementation of multiple malaria control tools. Gains made would however need to be sustained through continuous monitoring to ensure malaria elimination and eradication. Entomological inoculation rate (EIR) is currently the standard tool for transmission monitoring but this is not sensitive enough, especially in areas of very low transmission. Transmission estimation models based on seroconversion rates (λ) of antibodies to Plasmodium falciparum blood stage antigens are gaining relevance. Estimates of λ, which is the measure of transmission intensity, correlate with EIR but are limited by long-term persistence of antibodies to blood stage antigens. Seroprevalence of antibodies to sporozoite antigens may be better alternatives since these antigens usually have shorter immune exposure times. The aim of this study was to develop transmission estimation models based on the seroprevalence of antibodies to two P. falciparum sporozoite antigens (CSP, CelTOS) and compare with models based on the classical blood stage antigen AMA1., Methods: Antibody levels in archived plasma from three cross-sectional surveys conducted in 2009 in a low transmission area of Southern Ghana were assessed by indirect ELISA. Seroprevalence of antibodies against CSP, CelTOS and AMA1 were fitted to reversible catalytic models to estimate λ and corresponding seroreversion rates (ρ) for each antibody., Results: Of the three models developed, the anti-CSP model predicted a 13-fold decrease in λ four years prior to the time of sampling (2009). Anti-AMA1 antibodies formed at a four-fold greater rate compared to that of anti-CelTOS antibodies, and anti-CSP antibodies during the period of decreased λ. In contrast, anti-AMA1 antibodies decayed at a five-fold slower rate relative to that of anti-CSP antibodies while anti-AMA1 and anti-CelTOS antibody decay rates were not significantly different. Anti-CSP antibodies were relatively short-lived as they formed at an 11.6-fold slower rate relative to their decay during the period of decreased λ., Conclusions: These features of anti-CSP antibodies can be exploited for the development of models for predicting seasonal, short-term changes in transmission intensity in malaria-endemic areas, especially as the elimination phase of malaria control is approached.

Background: Malaria is responsible for up to a 600,000 deaths per year; conveying an urgent need for the development of a malaria vaccine. Studies with whole sporozoite vaccines in mice and non-human primates have shown that sporozoite-induced CD8+ T cells targeting liver stage antigens can mediate sterile protection. There is a need for a direct method to identify and phenotype malaria vaccine-induced CD8+ T cells in humans., Methods: Fluorochrome-labelled tetramers consisting of appropriate MHC class I molecules in complex with predicted binding peptides derived from Plasmodium falciparum AMA-1 were used to label ex vivo AMA-1 epitope specific CD8+ T cells from research subjects responding strongly to immunization with the NMRC-M3V-Ad-PfCA (adenovirus-vectored) malaria vaccine. The identification of these CD8+ T cells on the basis of their expression of early activation markers was also investigated., Results: Analyses by flow cytometry demonstrated that two of the six tetramers tested: TLDEMRHFY: HLA-A*01:01 and NEVVVKEEY: HLA-B*18:01, labelled tetramer-specific CD8+ T cells from two HLA-A*01:01 volunteers and one HLA-B*18:01 volunteer, respectively. By contrast, post-immune CD8+ T cells from all six of the immunized volunteers exhibited enhanced expression of the CD38 and HLA-DRhi early activation markers. For the three volunteers with positive tetramer staining, the early activation phenotype positive cells included essentially all of the tetramer positive, malaria epitope- specific CD8+ T cells suggesting that the early activation phenotype could identify all malaria vaccine-induced CD8+ T cells without prior knowledge of their exact epitope specificity., Conclusions: The results demonstrated that class I tetramers can identify ex vivo malaria vaccine antigen-specific CD8+ T cells and could therefore be used to determine their frequency, cell surface phenotype and transcription factor usage. The results also demonstrated that vaccine antigen-specific CD8+ T cells could be identified by activation markers without prior knowledge of their antigen-specificity, using a subunit vaccine for proof-of-concept. Whether, whole parasite or adjuvanted protein vaccines will also induce {CD38 and HLA-DRhi}+ CD8+ T cell populations reflective of the antigen-specific response will the subject of future investigations.

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.

Background: To prepare field sites for malaria vaccine trials, it is important to determine baseline antibody and T cell responses to candidate malaria vaccine antigens. Assessing T cell responses is especially challenging, given genetic restriction, low responses observed in endemic areas, their variability over time, potential suppression by parasitaemia and the intrinsic variability of the assays., Methods: In Part A of this study, antibody titres were measured in adults from urban and rural communities in Ghana to recombinant Plasmodium falciparum CSP, SSP2/TRAP, LSA1, EXP1, MSP1, MSP3 and EBA175 by ELISA, and to sporozoites and infected erythrocytes by IFA. Positive ELISA responses were determined using two methods. T cell responses to defined CD8 or CD4 T cell epitopes from CSP, SSP2/TRAP, LSA1 and EXP1 were measured by ex vivo IFN-γ ELISpot assays using HLA-matched Class I- and DR-restricted synthetic peptides. In Part B, the reproducibility of the ELISpot assay to CSP and AMA1 was measured by repeating assays of individual samples using peptide pools and low, medium or high stringency criteria for defining positive responses, and by comparing samples collected two weeks apart., Results: In Part A, positive antibody responses varied widely from 17%-100%, according to the antigen and statistical method, with blood stage antigens showing more frequent and higher magnitude responses. ELISA titres were higher in rural subjects, while IFA titres and the frequencies and magnitudes of ex vivo ELISpot activities were similar in both communities. DR-restricted peptides showed stronger responses than Class I-restricted peptides. In Part B, the most stringent statistical criteria gave the fewest, and the least stringent the most positive responses, with reproducibility slightly higher using the least stringent method when assays were repeated. Results varied significantly between the two-week time-points for many participants., Conclusions: All participants were positive for at least one malaria protein by ELISA, with results dependent on the criteria for positivity. Likewise, ELISpot responses varied among participants, but were relatively reproducible by the three methods tested, especially the least stringent, when assays were repeated. However, results often differed between samples taken two weeks apart, indicating significant biological variability over short intervals.

Background: Despite years of effort, a licensed malaria vaccine is not yet available. One of the obstacles facing the development of a malaria vaccine is the extensive heterogeneity of many of the current malaria vaccine antigens. To counteract this antigenic diversity, an effective malaria vaccine may need to elicit an immune response against multiple malaria antigens, thereby limiting the negative impact of variability in any one antigen. Since most of the malaria vaccine antigens that have been evaluated in people have not elicited a protective immune response, there is a need to identify additional protective antigens. In this study, the efficacy of three pre-erythrocytic stage malaria antigens was evaluated in a Plasmodium yoelii/mouse protection model., Methods: Mice were immunized with plasmid DNA and vaccinia virus vectors that expressed one, two or all three P. yoelii vaccine antigens. The immunized mice were challenged with 300 P. yoelii sporozoites and evaluated for subsequent infection., Results: Vaccines that expressed any one of the three antigens did not protect a high percentage of mice against a P. yoelii challenge. However, vaccines that expressed all three antigens protected a higher percentage of mice than a vaccine that expressed PyCSP, the most efficacious malaria vaccine antigen. Dissection of the multi-antigen vaccine indicated that protection was primarily associated with two of the three P. yoelii antigens. The protection elicited by a vaccine expressing these two antigens exceeded the sum of the protection elicited by the single antigen vaccines, suggesting a potential synergistic interaction., Conclusions: This work identifies two promising malaria vaccine antigen candidates and suggests that a multi-antigen vaccine may be more efficacious than a single antigen vaccine., (© 2011 Limbach et al; licensee BioMed Central Ltd.)