Blood-stage Vaccine Candidate Antigen Research Articles

IgG and IgM responses to the Plasmodium falciparum asexual stage antigens reflect respectively protection against malaria during pregnancy and infanthood.

Plasmodium falciparum malaria is a public health issue mostly seen in tropical countries. Until now, there is no effective malaria vaccine against antigens specific to the blood-stage of P. falciparum infection. Because the pathogenesis of malarial disease results from blood-stage infection, it is essential to identify the most promising blood-stage vaccine candidate antigens under natural exposure to malaria infection. A cohort of 400 pregnant women and their infants was implemented in South Benin. An active and passive protocol of malaria surveillance was established during pregnancy and infancy to precisely ascertain malaria infections during the follow-up. Twenty-eight antibody (Ab) responses specific to seven malaria candidate vaccine antigens were repeatedly quantified during pregnancy (3 time points) and infancy (6 time points) in order to study the Ab kinetics and their protective role. Abs were quantified by ELISA and logistic, linear and cox-proportional hazard model were performed to analyse the associations between Ab responses and protection against malaria in mothers and infants, taking into account socio-economic factors and for infants an environmental risk of exposure. The levels of IgM against MSP1, MSP2 and MSP3 showed an early protective response against the onset of symptomatic malaria infections starting from the 18th month of life, whereas no association was found for IgG responses during infancy. In women, some IgG responses tend to be associated with a protection against malaria risk along pregnancy and at delivery, among them IgG3 against GLURP-R0 and IgG2 against MSP1. The main finding suggests that IgM should be considered in vaccine designs during infanthood. Investigation of the functional role played by IgM in malaria protection needs further attention.

Characterization of a Plasmodium falciparum PHISTc protein, PF3D7_0801000, in blood- stage malaria parasites.

During intraerythrocytic development Plasmodium falciparum deploys numerous proteins to support erythrocyte invasion, intracellular growth and development, as well as host immune evasion. Since these proteins are key for parasite intraerythrocytic survival and propagation, they represent attractive targets for antimalarial vaccines. In this study we sought to characterize a member of the PHISTc family of proteins, PF3D7_0801000, as a potential vaccine target. Using the wheat germ cell-free system we expressed the N-terminal region of PF3D7_0801000 (G93-L494, PF3D7_0801000N) and generated specific immune sera. We observed that PF3D7_0801000 localizes in merozoites, and antibodies against PF3D7_0801000N modestly inhibit P. falciparum parasite growth in in vitro culture. Sliding window analysis of the coding sequence revealed that pf3d7_0801000n is relatively conserved among African parasite isolates. Antibody profiles in a malaria-exposed Ugandan population revealed that PF3D7_0801000N is strongly immunoreactive with antibody acquisition increasing with age. Taken together, these findings suggest the need for further evaluation of PF3D7_0801000 for its role in merozoite invasion and utility as an asexual blood-stage vaccine candidate antigen.

Changing pattern of the genetic diversities of Plasmodium falciparum merozoite surface protein-1 and merozoite surface protein-2 in Myanmar isolates

BackgroundPlasmodium falciparum merozoite surface protein-1 (PfMSP-1) and -2 (PfMSP-2) are major blood-stage vaccine candidate antigens. Understanding the genetic diversity of the genes, pfmsp-1 and pfmsp-2, is important for recognizing the genetic structure of P. falciparum, and the development of an effective vaccine based on the antigens. In this study, the genetic diversities of pfmsp-1 and pfmsp-2 in the Myanmar P. falciparum were analysed.MethodsThe pfmsp-1 block 2 and pfmsp-2 block 3 regions were amplified by polymerase chain reaction from blood samples collected from Myanmar patients who were infected with P. falciparum in 2013–2015. The amplified gene fragments were cloned into a T&A vector, and sequenced. Sequence analysis of Myanmar pfmsp-1 block 2 and pfmsp-2 block 3 was performed to identify the genetic diversity of the regions. The temporal genetic changes of both pfmsp-1 and pfmsp-2 in the Myanmar P. falciparum population, as well as the polymorphic diversity in the publicly available global pfmsp-1 and pfmsp-2, were also comparatively analysed.ResultsHigh levels of genetic diversity of pfmsp-1 and pfmsp-2 were observed in the Myanmar P. falciparum isolates. Twenty-eight different alleles of pfmsp-1 (8 for K1 type, 14 for MAD20 type, and 6 for RO33 type) and 59 distinct alleles of pfmsp-2 (18 for FC27, and 41 for 3D7 type) were identified in the Myanmar P. falciparum population in amino acid level. Comparative analyses of the genetic diversity of the Myanmar pfmsp-1 and pfmsp-2 alleles in the recent (2013–2015) and past (2004–2006) Myanmar P. falciparum populations indicated the dynamic genetic expansion of the pfmsp-1 and pfmsp-2 in recent years, suggesting that a high level of genetic differentiation and recombination of the two genes may be maintained. Population genetic structure analysis of the global pfmsp-1 and pfmsp-2 also suggested that a high level of genetic diversity of the two genes was found in the global P. falciparum population.ConclusionDespite the recent remarkable decline of malaria cases, the Myanmar P. falciparum population still remains of sufficient size to allow the generation and maintenance of genetic diversity. The high level of genetic diversity of pfmsp-1 and pfmsp-2 in the global P. falciparum population emphasizes the necessity for continuous monitoring of the genetic diversity of the genes for better understanding of the genetic make-up and evolutionary aspect of the genes in the global P. falciparum population.

Antibodies against a Plasmodium falciparum RON12 inhibit merozoite invasion into erythrocytes

Proteins coating Plasmodium merozoite surface and secreted from its apical organelles are considered as promising vaccine candidates for blood-stage malaria. The rhoptry neck protein 12 of Plasmodium falciparum (PfRON12) was recently reported as a protein specifically expressed in schizonts and localized to the rhoptry neck of merozoites. Here, we assessed its potential as a vaccine candidate. We expressed a recombinant PfRON12 protein by a wheat germ cell-free system to obtain anti-PfRON12 antibody. Immunoblot analysis of schizont lysates detected a single band at approximately 40 kDa under reducing conditions, consistent with the predicted molecular weight. Additionally, anti-PfRON12 antibody recognized a single band around 80 kDa under non-reducing conditions, suggesting native PfRON12 forms a disulfide-bond-mediated multimer. Immunofluorescence assay and immunoelectron microscopy revealed that PfRON12 localized to the rhoptry neck of merozoites in schizonts and to the surface of free merozoites. The biological activity of anti-PfRON12 antibody was tested by in vitro growth inhibition assay (GIA), and the rabbit antibodies significantly inhibited merozoite invasion of erythrocytes. We then investigated whether PfRON12 is immunogenic in P. falciparum-infected individuals. The sera from P. falciparum infected individuals in Thailand and Mali reacted with the recombinant PfRON12. Furthermore, human anti-PfRON12 antibodies affinity-purified from Malian serum samples inhibited merozoite invasion of erythrocytes in vitro. Moreover, pfron12 is highly conserved with only 4 non-synonymous mutations in the coding sequence from approximately 200 isolates deposited in PlasmoDB. These results suggest that PfRON12 might be a potential blood-stage vaccine candidate antigen against P. falciparum.

Functional Analysis Reveals Geographical Variation in Inhibitory Immune Responses Against a Polymorphic Malaria Antigen.

Plasmodium falciparum reticulocyte-binding protein homologue 2b (PfRh2b) is an invasion ligand that is a potential blood-stage vaccine candidate antigen; however, a naturally occurring deletion within an immunogenic domain is present at high frequencies in Africa and has been associated with alternative invasion pathway usage. Standardized tools that provide antigenic specificity in in vitro assays are needed to functionally assess the neutralizing potential of humoral responses against malaria vaccine candidate antigens. Transgenic parasite lines were generated to express the PfRh2b deletion. Total immunoglobulin G (IgG) from individuals residing in malaria-endemic regions in Tanzania, Senegal, and Mali were used in growth inhibition assays with transgenic parasite lines. While the PfRh2b deletion transgenic line showed no change in invasion pathway utilization compared to the wild-type in the absence of specific antibodies, it outgrew wild-type controls in competitive growth experiments. Inhibition differences with total IgG were observed in the different endemic sites, ranging from allele-specific inhibition to allele-independent inhibitory immune responses. The PfRh2b deletion may allow the parasite to escape neutralizing antibody responses in some regions. This difference in geographical inhibition was revealed using transgenic methodologies, which provide valuable tools for functionally assessing neutralizing antibodies against vaccine-candidate antigens in regions with varying malaria endemicity.

PfRON3 is an erythrocyte-binding protein and a potential blood-stage vaccine candidate antigen

BackgroundErythrocyte invasion by merozoites is an essential step in Plasmodium falciparum infection and leads to subsequent disease pathology. Proteins both on the merozoite surface and secreted from the apical organelles (micronemes, rhoptries and dense granules) mediate the invasion of erythrocytes; some of the molecules have been regarded as targets in the development of an anti-malaria vaccine. Recently, a subgroup of rhoptry neck proteins (PfRON2, PfRON4 and PfRON5) associated with the microneme protein apical membrane antigen AMA1 has been described as components of the moving junction complex that assists merozoite invasion into erythrocytes. However, unlike PfRON2, PfRON4 and PfRON5, the latest study suggested that PfRON3 might be located in the rhoptry bulb and participates in a novel PfRON complex (PfRON2, 3 and 4), but does not form a complex with AMA1. Additionally, the full-length PfRON3 protein possesses three transmembrane regions at the N-terminus, which is highly conserved among RON3 orthologues in the genus Plasmodium, Toxoplasma gondii and Eimeria tenella. Overall, these findings suggest that PfRON3 may play an important role in merozoite invasion into erythrocytes.ResultsPfRON3 was primarily expressed during the late trophozoite stage, with a peak in transcription levels at 40 hours post-invasion. The subcellular localization of PfRON3 was confirmed that it is a merozoite rhoptry bulb protein. Additionally, the recombinant form of PfRON3 protein bound to the erythrocyte and was recognized by sera collected from malaria endemic areas in Africa, and anti-PfRON3 antibodies significantly inhibited merozoite invasion into erythrocytes.MethodsThe expression of PfRON3 was analysed via real-time quantitative PCR, and the recombinant PfRON3 proteins were generated with an Escherichia coli expression system. The subcellular localization of PfRON3 was assessed with immunoelectron microscopy and immunofluorescence assay (IFA). The recognition PfRON3 by malaria immune sera was analysed with an enzyme-linked immunosorbent assay (ELISA). Erythrocyte-binding assays were performed using recombinant PfRON3 proteins and invasion inhibition assays were carried out with PfRON3-specific antibodies.ConclusionThis study confirmed that PfRON3 is a rhoptry protein with an erythrocyte-binding property, which is likely associated red blood cell invasion. PfRON3 is a potential vaccine candidate.Electronic supplementary materialThe online version of this article (doi:10.1186/1475-2875-13-490) contains supplementary material, which is available to authorized users.

Population genetics and natural selection in the gene encoding the Duffy binding protein II in Iranian Plasmodium vivax wild isolates

Region II of Duffy binding protein (PvDBP-II) is one of the most promising blood-stage vaccine candidate antigens against Plasmodium vivax and having knowledge of the nature and genetic polymorphism of PvDBP-II among global P. vivax isolates is important for developing a DBP-based vaccine. By using PCR and sequencing, the present molecular population genetic approach was carried out to investigate sequence diversity and natural selection of dbp-II gene in 63 P. vivax isolates collected from unstable and low transmission malaria-endemic areas of Iran during 2008–2012. Also, phylogenetic analysis, the diversifying natural selection, and recombination across the pvdbp-II gene, including regions containing B-cell epitopes were analyzed using the DnaSP and MEGA4 programs. Twenty two single nucleotide polymorphisms (SNPs, including 20 non-synonymous and 2 synonymous) were identified in PvDBP-II, resulting in 16 different PvDBP-II haplotypes among the Iranian P. vivax isolates. High binding inhibitory B-cell epitope (H3) overlapping with intrinsically unstructured/disordered region (aa: 384–392) appeared to be highly polymorphic (D384G/E385K/ K386N/Q/R390H), and positive selective pressure acted on this region. Most of the polymorphic amino acids, which are located on the surface of the protein, are under selective pressure that implies increased recombination events and exposure to the human immune system.In summary, PvDBP-II gene displays genetic polymorphism among Iranian P. vivax isolates and it is under selective pressure. Mutations, recombination, and positive selection seem to play a role in the resulting genetic diversity, and phylogenetic analysis of DNA sequences demonstrates that Iranian isolates represent a sample of the global population. These results are useful for understanding the nature of the P. vivax population in Iran and also for development of PvDBP-II-based malaria vaccine.

Assessment of antibody-dependent respiratory burst activity from mouse neutrophils on Plasmodium yoelii malaria challenge outcome.

New tools are required to expedite the development of an effective vaccine against the blood-stage infection with the human malaria parasite Plasmodium falciparum. This work describes the assessment of the ADRB assay in a mouse model, characterizing the functional interaction between antimalarial serum antibodies and FcRs upon neutrophils. We describe a reproducible, antigen-specific assay, dependent on functional FcR signaling, and show that ADRB activity is induced equally by IgG1 and IgG2a isotypes and is modulated by blocking FcR function. However, following immunization of mice with the blood-stage vaccine candidate antigen MSP142, no measurable ADRB activity was induced against PEMS and neither was vaccine efficacy modulated against Plasmodium yoelii blood-stage challenge in γ(-/-) mice compared with WT mice. In contrast, following a primary, nonlethal P. yoelii parasite challenge, serum from vaccinated mice and nonimmunized controls showed anti-PEMS ADRB activity. Upon secondary challenge, nonimmunized γ(-/-) mice showed a reduced ability to control blood-stage parasitemia compared with immunized γ(-/-) mice; however, WT mice, depleted of their neutrophils, did not lose their ability to control infection. Thus, whereas neutrophil-induced ADRB against PEMS does not appear to play a role in protection against P. yoelii rodent malaria, induction of ADRB activity after challenge suggests that antigen targets of anti-PEMS ADRB activity remain to be established, as well as further supporting the observation that ADRB activity to P. falciparum arises following repeated natural exposure.

RALP1 Is a Rhoptry Neck Erythrocyte-Binding Protein of Plasmodium falciparum Merozoites and a Potential Blood-Stage Vaccine Candidate Antigen

Erythrocyte invasion by merozoites is an obligatory stage of Plasmodium infection and is essential to disease progression. Proteins in the apical organelles of merozoites mediate the invasion of erythrocytes and are potential malaria vaccine candidates. Rhoptry-associated, leucine zipper-like protein 1 (RALP1) of Plasmodium falciparum was previously found to be specifically expressed in schizont stages and localized to the rhoptries of merozoites by immunofluorescence assay (IFA). Also, RALP1 has been refractory to gene knockout attempts, suggesting that it is essential for blood-stage parasite survival. These characteristics suggest that RALP1 can be a potential blood-stage vaccine candidate antigen, and here we assessed its potential in this regard. Antibodies were raised against recombinant RALP1 proteins synthesized by using the wheat germ cell-free system. Immunoelectron microscopy demonstrated for the first time that RALP1 is a rhoptry neck protein of merozoites. Moreover, our IFA data showed that RALP1 translocates from the rhoptry neck to the moving junction during merozoite invasion. Growth and invasion inhibition assays revealed that anti-RALP1 antibodies inhibit the invasion of erythrocytes by merozoites. The findings that RALP1 possesses an erythrocyte-binding epitope in the C-terminal region and that anti-RALP1 antibodies disrupt tight-junction formation, are evidence that RALP1 plays an important role during merozoite invasion of erythrocytes. In addition, human sera collected from areas in Thailand and Mali where malaria is endemic recognized this protein. Overall, our findings indicate that RALP1 is a rhoptry neck erythrocyte-binding protein and that it qualifies as a potential blood-stage vaccine candidate.

Antibodies by Non-Febrile, Smear-Negative Individuals from a Malaria Epidemic-Prone Setting in Ethiopia are Strongly Reactive to Plasmodium falciparum Blood-Stage Vaccine Candidate Antigens

Background: Plasmodium falciparum malaria remains a major public health concern globally though there is some decline due to scale-up of control efforts. Evaluation of the anti-malarial immune profile, in populations residing in epidemic-prone areas in the dry season or at the time when vector control largely reduced man-mosquito contact, would help predict malaria burden when future epidemics occur. Methods: A cross-sectional study was designed to investigate antibody responses to four P. falciparum blood stage vaccine candidate antigens in non-febrile individuals from Shewa Robit in north-central Ethiopia where malaria transmission was expected at a minimal level as a result of the sampling season and effective vector control. Blood samples were analyzed microscopically for Plasmodium detection. The enzyme-linked immunosorbent assay (ELISA) was used to measure immunoglobulin (IgG) antibodies to apical membrane antigen 1 (AMA1), glutamaterich protein (GLURP) R2 region and merozoite surface protein 2 (MSP2) allelic variants. Results: Study participants were smear-negative for Plasmodium infection. While 51 (22%) of the participants reported that they had never been exposed to clinical malaria in life, 177 (78%) reported at least one clinical malaria episode with laboratory confirmed P. falciparum infection. The antigens tested were well-recognized by the test sera although significant differences were observed in antibody prevalence and level between the different antigens and there was inter-individual variability. IgG response to the antigens showed age-related pattern but without evidence of relation with status and frequency of reported past exposure to clinical malaria. Conclusion: The data suggests that individuals in an epidemic-prone malaria setting have reactive and stable antibodies that readily recognize P. falciparum blood-stage vaccine candidate antigens in the absence of slidepositivity. Analysis of age-related pattern in antibody level showed positive association with age but unrelated with increasing frequency of reported episode suggesting the role of intrinsic age-related factors in malaria immune maturation.

Genetic diversity and natural selection of Duffy binding protein of Plasmodium vivax Korean isolates

Plasmodium vivax Duffy binding protein (PvDBP) is a micronemal type I membrane protein that plays an essential role in erythrocyte invasion of merozoites. PvDBP is a prime blood stage vaccine candidate antigen against P. vivax, but its polymorphic nature represents a major obstacle to the successful design of a protective vaccine against vivax malaria. In this study, we analyzed the genetic polymorphism and natural selection at the N-terminal cysteine-rich region of PvDBP (PvDBPII) among 70 P. vivax isolates collected from Korean patients during 2005–2010. Seventeen single nucleotide polymorphisms (SNP), which resulted in 14 non-synonymous and 3 synonymous mutations, were found in PvDBPII among the Korean P. vivax isolates. Sequence analyses revealed that 13 different PvDBPII haplotypes, which were clustered into 3 distinct clades, were identified in Korean P. vivax isolates. The difference between the rates of nonsynomyous and synonymous mutations suggested that the region has evolved under natural selection. High selective pressure preferentially acted on regions identified or predicted to be B- and T-cell epitopes and MHC binding regions of PvDBPII. Recombination may also contribute to genetic diversity of PvDBPII. Our results suggest that PvDBPII of Korean P. vivax isolates display a limited genetic polymorphism and are under selective pressure. These results have significant implications for understanding the nature of the P. vivax population circulating in Korea and provide useful information for development of malaria vaccines based on this antigen.

Genetic polymorphism and natural selection of Duffy binding protein of Plasmodium vivax Myanmar isolates

BackgroundPlasmodium vivax Duffy binding protein (PvDBP) plays an essential role in erythrocyte invasion and a potential asexual blood stage vaccine candidate antigen against P. vivax. The polymorphic nature of PvDBP, particularly amino terminal cysteine-rich region (PvDBPII), represents a major impediment to the successful design of a protective vaccine against vivax malaria. In this study, the genetic polymorphism and natural selection at PvDBPII among Myanmar P. vivax isolates were analysed.MethodsFifty-four P. vivax infected blood samples collected from patients in Myanmar were used. The region flanking PvDBPII was amplified by PCR, cloned into Escherichia coli, and sequenced. The polymorphic characters and natural selection of the region were analysed using the DnaSP and MEGA4 programs.ResultsThirty-two point mutations (28 non-synonymous and four synonymous mutations) were identified in PvDBPII among the Myanmar P. vivax isolates. Sequence analyses revealed that 12 different PvDBPII haplotypes were identified in Myanmar P. vivax isolates and that the region has evolved under positive natural selection. High selective pressure preferentially acted on regions identified as B- and T-cell epitopes of PvDBPII. Recombination may also be played a role in the resulting genetic diversity of PvDBPII.ConclusionsPvDBPII of Myanmar P. vivax isolates displays a high level of genetic polymorphism and is under selective pressure. Myanmar P. vivax isolates share distinct types of PvDBPII alleles that are different from those of other geographical areas. These results will be useful for understanding the nature of the P. vivax population in Myanmar and for development of PvDBPII-based vaccine.

Genetic polymorphism and natural selection of Duffy binding protein of Plasmodium vivax Myanmar isolates

BackgroundPlasmodium vivax Duffy binding protein (PvDBP) plays an essential role in erythrocyte invasion and a potential asexual blood stage vaccine candidate antigen against P. vivax. The polymorphic nature of PvDBP, particularly amino terminal cysteine-rich region (PvDBPII), represents a major impediment to the successful design of a protective vaccine against vivax malaria. In this study, the genetic polymorphism and natural selection at PvDBPII among Myanmar P. vivax isolates were analysed.MethodsFifty-four P. vivax infected blood samples collected from patients in Myanmar were used. The region flanking PvDBPII was amplified by PCR, cloned into Escherichia coli, and sequenced. The polymorphic characters and natural selection of the region were analysed using the DnaSP and MEGA4 programs.ResultsThirty-two point mutations (28 non-synonymous and four synonymous mutations) were identified in PvDBPII among the Myanmar P. vivax isolates. Sequence analyses revealed that 12 different PvDBPII haplotypes were identified in Myanmar P. vivax isolates and that the region has evolved under positive natural selection. High selective pressure preferentially acted on regions identified as B- and T-cell epitopes of PvDBPII. Recombination may also be played a role in the resulting genetic diversity of PvDBPII.ConclusionsPvDBPII of Myanmar P. vivax isolates displays a high level of genetic polymorphism and is under selective pressure. Myanmar P. vivax isolates share distinct types of PvDBPII alleles that are different from those of other geographical areas. These results will be useful for understanding the nature of the P. vivax population in Myanmar and for development of PvDBPII-based vaccine.

Discovery of GAMA, a Plasmodium falciparum Merozoite Micronemal Protein, as a Novel Blood-Stage Vaccine Candidate Antigen

One of the solutions for reducing the global mortality and morbidity due to malaria is multivalent vaccines comprising antigens of several life cycle stages of the malarial parasite. Hence, there is a need for supplementing the current set of malaria vaccine candidate antigens. Here, we aimed to characterize glycosylphosphatidylinositol (GPI)-anchored micronemal antigen (GAMA) encoded by the PF08_0008 gene in Plasmodium falciparum. Antibodies were raised against recombinant GAMA synthesized by using a wheat germ cell-free system. Immunoelectron microscopy demonstrated for the first time that GAMA is a microneme protein of the merozoite. Erythrocyte binding assays revealed that GAMA possesses an erythrocyte binding epitope in the C-terminal region and it binds a nonsialylated protein receptor on human erythrocytes. Growth inhibition assays revealed that anti-GAMA antibodies can inhibit P. falciparum invasion in a dose-dependent manner and GAMA plays a role in the sialic acid (SA)-independent invasion pathway. Anti-GAMA antibodies in combination with anti-erythrocyte binding antigen 175 exhibited a significantly higher level of invasion inhibition, supporting the rationale that targeting of both SA-dependent and SA-independent ligands/pathways is better than targeting either of them alone. Human sera collected from areas of malaria endemicity in Mali and Thailand recognized GAMA. Since GAMA in P. falciparum is refractory to gene knockout attempts, it is essential to parasite invasion. Overall, our study indicates that GAMA is a novel blood-stage vaccine candidate antigen.

Plasmodium berghei Circumvents Immune Responses Induced by Merozoite Surface Protein 1- and Apical Membrane Antigen 1-Based Vaccines

BackgroundTwo current leading malaria blood-stage vaccine candidate antigens for Plasmodium falciparum, the C-terminal region of merozoite surface protein 1 (MSP119) and apical membrane antigen 1 (AMA1), have been prioritized because of outstanding protective efficacies achieved in a rodent malaria Plasmodium yoelii model. However, P. falciparum vaccines based on these antigens have had disappointing outcomes in clinical trials. Discrepancies in the vaccine efficacies observed between the P. yoelii model and human clinical trials still remain problematic.Methodology and ResultsIn this study, we assessed the protective efficacies of a series of MSP119- and AMA1-based vaccines using the P. berghei rodent malarial parasite and its transgenic models. Immunization of mice with a baculoviral-based vaccine (BBV) expressing P. falciparum MSP119 induced high titers of PfMSP119-specific antibodies that strongly reacted with P. falciparum blood-stage parasites. However, no protection was achieved following lethal challenge with transgenic P. berghei expressing PfMSP119 in place of native PbMSP119. Similarly, neither P. berghei MSP119- nor AMA1-BBV was effective against P. berghei. In contrast, immunization with P. yoelii MSP119- and AMA1-BBVs provided 100% and 40% protection, respectively, against P. yoelii lethal challenge. Mice that naturally acquired sterile immunity against P. berghei became cross-resistant to P. yoelii, but not vice versa.ConclusionThis is the first study to address blood-stage vaccine efficacies using both P. berghei and P. yoelii models at the same time. P. berghei completely circumvents immune responses induced by MSP119- and AMA1-based vaccines, suggesting that P. berghei possesses additional molecules and/or mechanisms that circumvent the host's immune responses to MSP119 and AMA1, which are lacking in P. yoelii. Although it is not known whether P. falciparum shares these escape mechanisms with P. berghei, P. berghei and its transgenic models may have potential as useful tools for identifying and evaluating new blood-stage vaccine candidate antigens for P. falciparum.

Duration of Naturally Acquired Antibody Responses to Blood-StagePlasmodium falciparumIs Age Dependent and Antigen Specific

Naturally acquired antibody responses provide partial protection from clinical malaria, and blood-stage parasite vaccines under development aim to prime such responses. To investigate the determinants of antibody response longevity, immunoglobulin G (IgG) antibodies to several blood-stage vaccine candidate antigens in the sera of two cohorts of children of up to 6 years of age during the dry seasons of 2003 and 2004 in The Gambia were examined. The first cohort showed that most antibodies were lost within less than 4 months of the first sampling if a persistent infection was not present, so the study of the second-year cohort involved collecting samples from individuals every 2 weeks over a 3-month period. Antibody responses in the second cohort were also influenced by persistent malaria infection, so analysis focused particularly on children in whom parasites were not detected after the first time point. Antibodies to most antigens declined more slowly in children in the oldest age group (>5 years old) and more rapidly in children in the youngest group (<3 years old). However, antibodies to merozoite surface protein 2 were shorter lived than antibodies to other antigens and were not more persistent in older children. The age-specific and antigen-specific differences were not explained by different IgG subclass response profiles, indicating the probable importance of differential longevities of plasma cell populations rather than antibody molecules. It is likely that young children mostly have short-lived plasma cells and thus experience rapid declines in antibody levels but that older children have longer-lasting antibody responses that depend on long-lived plasma cells.

Cellular and humoral immune responses to well-defined blood stage antigens (major merozoite surface antigen) of Plasmodium falciparum in adults from an Indian zone where malaria is endemic.

Conserved and variant regions of two blood stage vaccine candidate antigens of Plasmodium falciparum, merozoite surface antigen (MSA-1) and ring-infected erythrocyte surface antigen (Pf155/RESA), have been shown to be immunogenic. However, the relative immunogenicity of these immunogens in different populations has not been studied. The conserved N-terminal region of MSA-1 was investigated for its immunogenicity by studying cellular (T cell) and humoral (B cell) immune responses in P. falciparum-primed individuals, living in malaria-hyperendemic areas (Orissa State, India), where malaria presents an alarming situation. MSA-1-derived synthetic peptides contained sequences that activated T cells to proliferate and release gamma interferon in vitro. There was considerable variation in the responses to different peptides. However, the highest responses (51% [18 of 35] by proliferation and 34% [12 of 35] by gamma interferon release) were obtained with a synthetic hybrid peptide containing sequences from conserved N- and C-terminal repeat regions of MSA-1 and Pf155/RESA, respectively. Antibody reactivities in an enzyme immunoassay of plasma samples from these donors to different peptides used for T-cell activation were heterogeneous. In general, there was poor correlation between DNA synthesis and either gamma interferon release or antibody responses in individual donors, underlining the importance of examining several parameters of T-cell activation to assess the total T-cell responsiveness of a study population to a given antigen. However, the results from our studies suggest that synthetic constructs containing sequences from the N- and C-terminal regions of MSA-1 and Pf155/RESA representing different erythrocytic stages of the P. falciparum parasite are more immunogenic in humans living in malaria-hyperendemic areas of India who have been primed by natural infection.

Ion-exchange—immunoaffinity purification of a recombinant baculovirus Plasmodium falciparum apical membrane antigen, PF83/AMA-1

A two-step purification regime has been developed for a quantitatively minor, putatively transmembrane, M r 83 000, apical membrane blood stage vaccine candidate antigen of Plasmodium falciparum (PF83/AMA-1), that has been expressed as a full-length baculovirus recombinant protein, PF83-7G8-1. The first step utilizes a new approach to high-performance ion-exchange chromatography (HPIEC) in which elution conditions are not only defined by charge, but also by hydrophobicity. HPIEC fractionation involves successive sodium chloride gradient anion-exchange elutions (A and B), where a change in the non-ionic detergent polyoxyethylenealkylether C 10E 5 concentration between elutions A and B (from 0.01% to 0.1% (w/v) respectively), results in a fraction that comprises from 2% to 9% PF83-7G8-1. Subsequent column immunoaffinity purification of this fraction on Q-Sepharose CL 4B-28G2dc1 mAb yields a PF83-7G8- 1 preparation that is 56% pure. Rat mAb 28G2dc1 recognizes a C-terminal region that is conserved and cross reactive within the AMA-1 family, thus permitting recombinant and native full-length AMA-1 molecules from other species to be purified for molecular analysis. Immunological and molecular characterisation of the vaccine-related characteristics of purified PF83/AMA-1 are now underway.

Enzyme-linked staining method for light microscopic detection of antibodies to parasite antigens on the membrane of Plasmodium falciparum infected erythrocytes

Indirect erythrocyte membrane immunofluorescence (EMIF), a standard method for detection of antibodies to the P. falciparum blood stage vaccine candidate antigen Pf155/RESA, has been adapted to light microscopy by enzyme-linked immunostaining of the erythrocyte membrane, using alkaline phosphatase and chromogenic substrate. This method gives a dark blue staining of the membranes of ring infected erythrocytes. Results obtained with 70 African sera in EMIF and in enzyme immunostaining correlated well although the enzyme based method sometimes resulted in higher antibody titers and appeared to be slightly more sensitive. Similar results were obtained when comparing immunofluorescence with enzyme immunostaining for detection of antibodies to intraerythrocytic parasite antigens. The enzyme linked immunostaining described is simple and fast and does not require expensive equipment and should, thus, be well suited for use in laboratories with limited resources or under field conditions.