Towards a blood-stage vaccine for malaria: Are we following all the leads?

Although the malaria parasite was discovered more than 120 years ago, it is only during the past 20 years, following the cloning of malaria genes, that we have been able to think rationally about vaccine design and development. Effective vaccines for malaria could interrupt the life cycle of the parasite at different stages in the human host or in the mosquito. The purpose of this review is to outline the challenges we face in developing a vaccine that will limit growth of the parasite during the stage within red blood cells - the stage responsible for all the symptoms and pathology of malaria. More than 15 vaccine trials have either been completed or are in progress, and many more are planned. Success in current trials could lead to a vaccine capable of saving more than 2 million lives per year.

Immunity to asexual blood stage malaria and vaccine approaches

The development of a malaria vaccine seems to be a definite possibility despite the fact that even individuals with a life time of endemic exposure do not develop sterile immunity. An effective malaria vaccine would be invaluable in preventing malaria-associated deaths in endemic areas, especially amongst children less than 5 years of age and pregnant women. This review discusses our current understanding of immunity against the asexual blood stage of malaria - the stage that is responsible for the symptoms of the disease - and approaches to the design of an asexual blood stage vaccine.

Adapting immunity with subunit vaccines: case studies with group A Streptococcus and malaria

Although vaccines have widely been regarded as the most cost-effective way to improve public health, for some organisms new technological advances in vaccine design and delivery, incurring additional developmental costs, will be essential. These organisms are typically those for which natural immunity is either slow to develop or does not develop at all. Clearly, such organisms have evolved strategies to evade immune responses and innovative approaches will be required to induce a type of immune response which is both different to that which develops naturally and is effective. This article describes some approaches to develop vaccines for two such organisms (malaria parasites and Streptococcus pyogenes (group A Streptococcus)) that are associated with widespread mortality and morbidity, mostly in the poorest countries of the world. At this stage, the challenges are primarily scientific, but if these hurdles are surmounted then the challenges will become financial ones - developing much needed vaccines for people least able to afford them. (C) 2002 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.

Comparative recognition by human IgG antibodies of recombinant proteins representing three asexual erythrocytic stage vaccine candidates of Plasmodium vivax

In previous immuno-epidemiological studies of the naturally acquired antibody responses to merozoite surface protein-1 (MSP-1) of Plasmodium vivax, we had evidence that the responses to distinct erythrocytic stage antigens could be differentially regulated. The present study was designed to compare the antibody response to three asexual erythrocytic stage antigens vaccine candidates of P. vivax. Recombinant proteins representing the 19 kDa C-terminal region of MSP-1(PvMSP19), apical membrane antigen n-1 ectodomain (PvAMA-1), and the region II of duffy binding protein (PvDBP-RII) were compared in their ability to bind to IgG antibodies of serum samples collected from 220 individuals from the state of Pará, in the North of Brazil. During patent infection with P. vivax, the frequency of individuals with IgG antibodies to PvMSP1(19), PvAMA-1, and PvDBP-RII were 95, 72.7, and 44.5% respectively. Although the frequency of responders to PvDBP-RII was lower, this frequency increased in individuals following multiple malarial infections. Individually, the specific antibody levels did not decline significantly nine months after treatment, except to PvMSP1(19). Our results further confirm a complex regulation of the immune response to distinct blood stage antigens. The reason for that is presently unknown but it may contribute to the high risk of re-infection in individuals living in the endemic areas.

Acquired antibody responses against Plasmodium vivax infection vary with host genotype for duffy antigen receptor for chemokines (DARC).

BACKGROUND: Polymorphism of the Duffy Antigen Receptor for Chemokines (DARC) is associated with susceptibility to and the severity of Plasmodium vivax malaria in humans. P. vivax uses DARC to invade erythrocytes. Individuals lacking DARC are 'resistant' to P. vivax erythrocytic infection. However, susceptibility to P. vivax in DARC+ individuals is reported to vary between specific DARC genotypes. We hypothesized that the natural acquisition of antibodies to P. vivax blood stages may vary with the host genotype and the level of DARC expression. Furthermore, high parasitemia has been reported to effect the acquisition of immunity against pre-erythrocytic parasites. We investigated the correlation between host DARC genotypes and the frequency and magnitude of antibodies against P. vivax erythrocytic stage antigens. METHODOLOGY/FINDINGS: We assessed the frequencies and magnitudes of antibody responses against P. vivax and P. falciparum sporozoite and erythrocytic antigens in Colombian donors from malaria-endemic regions. The frequency and level of naturally-acquired antibodies against the P. vivax erythrocytic antigens merozoite surface protein 1 (PvMSP1) and Duffy binding protein (PvDBP) varied with the host DARC genotypes. Donors with one negative allele (FY*B/FY*Bnull and FY*A/FY*Bnull) were more likely to have anti-PvMSP1 and anti-PvDBP antibodies than those with two positive alleles (FY*B/FY*B and FY*A/FY*B). The lower IgG3 and IgG1 components of the total IgG response may account for the decreased responses to P. vivax erythrocytic antigens with FY*A/FY*B and FY*B/FY*B genotypes. No such association was detected with P. falciparum erythrocytic antigens, which does not use DARC for erythrocyte invasion. CONCLUSION/SIGNIFICANCE: Individuals with higher DARC expression, which is associated with higher susceptibility to P. vivax infection, exhibited low frequencies and magnitudes of P. vivax blood-stage specific antibody responses. This may indicate that one of the primary mechanisms by which P. vivax evades host immunity is through DARC indirectly down-regulating humoral responses against erythrocytic invasion and development.

Expression and one-step purification of Plasmodium proteins in dictyostelium.

Nearly full-length Circumsporozoite protein (CSP) from Plasmodium falciparum, the C-terminal fragments from both P. falciparm and P. yoelii CSP and a fragment comprising 351 amino acids of P.vivax MSPI were expressed in the slime mold Dictyostelium discoideum. Discoidin-tag expression vectors allowed both high yields of these proteins and their purification by a nearly single-step procedure. We exploited the galactose binding activity of Discoidin Ia to separate the fusion proteins by affinity chromatography on Sepharose-4B columns. Inclusion of a thrombin recognition site allowed cleavage of the Discoidin-tag from the fusion protein. Partial secretion of the protein was obtained via an ER independent pathway, whereas routing the recombinant proteins to the ER resulted in glycosylation and retention. Yields of proteins ranged from 0.08 to 3 mg l(-1) depending on the protein sequence and the purification conditions. The recognition of purified MSPI by sera from P. vivax malaria patients was used to confirm the native conformation of the protein expressed in Dictyostelium. The simple purification procedure described here, based on Sepharose-4B, should facilitate the expression and the large-scale purification of various Plasmodium polypeptides.

Antibody levels against GLURP R2, MSP1 block 2 hybrid and AS202.11 and the risk of malaria in children living in hyperendemic (Burkina Faso) and hypo-endemic (Ghana) areas.

Differences in parasite transmission intensity influence the process of acquisition of host immunity to Plasmodium falciparum malaria and ultimately, the rate of malaria related morbidity and mortality. Potential vaccines being designed to complement current intervention efforts therefore need to be evaluated against different malaria endemicity backgrounds. The associations between antibody responses to the chimeric merozoite surface protein 1 block 2 hybrid (MSP1 hybrid), glutamate-rich protein region 2 (GLURP R2) and the peptide AS202.11, and the risk of malaria were assessed in children living in malaria hyperendemic (Burkina Faso, n = 354) and hypo-endemic (Ghana, n = 209) areas. Using the same reagent lots and standardized protocols for both study sites, immunoglobulin (Ig) M, IgG and IgG sub-class levels to each antigen were measured by ELISA in plasma from the children (aged 6-72 months). Associations between antibody levels and risk of malaria were assessed using Cox regression models adjusting for covariates. There was a significant association between GLURP R2 IgG3 and reduced risk of malaria after adjusting age of children in both the Burkinabe (hazard ratio 0.82; 95 % CI 0.74-0.91, p < 0.0001) and the Ghanaian (HR 0.48; 95 % CI 0.25-0.91, p = 0.02) cohorts. MSP1 hybrid IgM was associated (HR 0.85; 95 % CI 0.73-0.98, p = 0.02) with reduced risk of malaria in Burkina Faso cohort while IgG against AS202.11 in the Ghanaian children was associated with increased risk of malaria (HR 1.29; 95 % CI 1.01-1.65, p = 0.04). These findings support further development of GLURP R2 and MSP1 block 2 hybrid, perhaps as a fusion vaccine antigen targeting malaria blood stage that can be deployed in areas of varying transmission intensity.

Development of designed site-directed pseudopeptide-peptido-mimetic immunogens as novel minimal subunit-vaccine candidates for malaria

Synthetic vaccines constitute the most promising tools for controlling and preventing infectious diseases. When synthetic immunogens are designed from the pathogen native sequences, these are normally poorly immunogenic and do not induce protection, as demonstrated in our research. After attempting many synthetic strategies for improving the immunogenicity properties of these sequences, the approach consisting of identifying high binding motifs present in those, and then performing specific changes on amino-acids belonging to such motifs, has proven to be a workable strategy. In addition, other strategies consisting of chemically introducing non-natural constraints to the backbone topology of the molecule and modifying the a-carbon asymmetry are becoming valuable tools to be considered in this pursuit. Non-natural structural constraints to the peptide backbone can be achieved by introducing peptide bond isosters such as reduced amides, partially retro or retro-inverso modifications or even including urea motifs. The second can be obtained by strategically replacing L-amino-acids with their enantiomeric forms for obtaining both structurally site-directed designed immunogens as potential vaccine candidates and their Ig structural molecular images, both having immunotherapeutic effects for preventing and controlling malaria.

Plasmodium vivax: Microsatellite analysis of multiple-clone infections

We used mixtures of genomic DNA from two genetically distinct isolates from Brazil, 42M and 312M, to investigate how accurately 12-locus microsatellite typing describes the overall genetic diversity and characterizes multilocus haplotypes in multiple-clone Plasmodium vivax infections. We found varying PCR amplification efficiencies of microsatellite alleles; for example, from the same 1:1 mixture of 42M and 312M DNA we amplified predominantly 312M-type alleles at 10 loci and 42M-type alleles at 2 loci. All microsatellite alleles were accurately scored in 1:0.5 and 1:0.25 312M:42M DNA mixtures, even when minor peak heights did not meet previously suggested criteria for minor allele detection in multiple-clone infections. Relative proportions of major and minor alleles were unaffected by multiple displacement amplification of template DNA prior to PCR-based microsatellite typing. Although microsatellite typing may detect minor alleles in clone mixtures, amplification biases may lead to inaccurate assignment of predominant haplotypes in multiple-clone P. vivax infections. (C) 2008 Elsevier Inc. All rights reserved.

Comparative recognition by human IgG antibodies of recombinant proteins representing three asexual erythrocytic stage vaccine candidates of Plasmodium vivax

In previous immuno-epidemiological studies of the naturally acquired antibody responses to merozoite surface protein-1 (MSP-1) of Plasmodium vivax, we had evidence that the responses to distinct erythrocytic stage antigens could be differentially regulated. The present study was designed to compare the antibody response to three asexual erythrocytic stage antigens vaccine candidates of P. vivax. Recombinant proteins representing the 19 kDa C-terminal region of MSP-1(PvMSP19), apical membrane antigen n-1 ectodomain (PvAMA-1), and the region II of duffy binding protein (PvDBP-RII) were compared in their ability to bind to IgG antibodies of serum samples collected from 220 individuals from the state of Pará, in the North of Brazil. During patent infection with P. vivax, the frequency of individuals with IgG antibodies to PvMSP119, PvAMA-1, and PvDBP-RII were 95, 72.7, and 44.5% respectively. Although the frequency of responders to PvDBP-RII was lower, this frequency increased in individuals following multiple malarial infections. Individually, the specific antibody levels did not decline significantly nine months after treatment, except to PvMSP119. Our results further confirm a complex regulation of the immune response to distinct blood stage antigens. The reason for that is presently unknown but it may contribute to the high risk of re-infection in individuals living in the endemic areas.

Extracellular ATP triggers proteolysis and cytosolic Ca2+ rise in Plasmodium berghei and Plasmodium yoelii malaria parasites

Background: Plasmodium has a complex cell biology and it is essential to dissect the cell-signalling pathways underlying its survival within the host. Methods: Using the fluorescence resonance energy transfer (FRET) peptide substrate Abz-AIKFFARQ-EDDnp and Fluo4/AM, the effects of extracellular ATP on triggering proteolysis and Ca2+ signalling in Plasmodium berghei and Plasmodium yoelii malaria parasites were investigated. Results: The protease activity was blocked in the presence of the purinergic receptor blockers suramin (50 mu M) and PPADS (50 mu M) or the extracellular and intracellular calcium chelators EGTA (5 mM) and BAPTA/AM (25, 100, 200 and 500 mu M), respectively for P. yoelii and P. berghei. Addition of ATP (50, 70, 200 and 250 mu M) to isolated parasites previously loaded with Fluo4/AM in a Ca2+-containing medium led to an increase in cytosolic calcium. This rise was blocked by pre-incubating the parasites with either purinergic antagonists PPADS (50 mu M), TNP-ATP (50 mu M) or the purinergic blockers KN-62 (10 mu M) and Ip5I (10 mu M). Incubating P. berghei infected cells with KN-62 (200 mu M) resulted in a changed profile of merozoite surface protein 1 (MSP1) processing as revealed by western blot assays. Moreover incubating P. berghei for 17 h with KN-62 (10 mu M) led to an increase in rings forms (82% +/- 4, n = 11) and a decrease in trophozoite forms (18% +/- 4, n = 11). Conclusions: The data clearly show that purinergic signalling modulates P. berghei protease(s) activity and that MSP1 is one target in this pathway.

Geographic differentiation of polymorphism in the Plasmodium falciparum malaria vaccine candidate gene SERA5

SERA5 is regarded as a promising malaria vaccine candidate of the most virulent human malaria parasite Plasmodium falciparum. SERA5 is a 120 kDa abundantly expressed blood-stage protein containing a papain-like protease. Since substantial polymorphism in blood-stage vaccine candidates may potentially limit their efficacy, it is imperative to fully investigate polymorphism of the SERA5 gene (sera5). In this study, we performed evolutionary and population genetic analysis of sera5. The level of inter-species divergence (kS = 0.076) between P. falciparum and Plasmodium reichenowi, a closely related chimpanzee malaria parasite is comparable to that of housekeeping protein genes. A signature of purifying selection was detected in the proenzyme and enzyme domains. Analysis of 445 near full-length P. falciparum sera5 sequences from nine countries in Africa, Southeast Asia, Oceania and South America revealed extensive variations in the number of octamer repeat (OR) and serine repeat (SR) regions as well as substantial level of single nucleotide polymorphism (SNP) in non-repeat regions (2562 bp). Remarkably, a 14 amino acid sequence of SERA5 (amino acids 59-72) that is known to be the in vitro target of parasite growth inhibitory antibodies was found to be perfectly conserved in all 445 worldwide isolates of P. falciparum evaluated. Unlike other major vaccine target antigen genes such as merozoite surface protein-1, apical membrane antigen-1 or circumsporozoite protein, no strong evidence for positive selection was detected for SNPs in the non-repeat regions of sera5. A biased geographical distribution was observed in SNPs as well as in the haplotypes of the sera5 OR and SR regions. In Africa, OR- and SR-haplotypes with low frequency (<5%) and SNPs with minor allele frequency (<5%) were abundant and were mostly continent-specific. Consistently, significant genetic differentiation, assessed by the Wright's fixation index (FST) of inter-population variance in allele frequencies, was detected for SNPs and both OR- and SR-haplotypes among almost all parasite populations. The exception was parasite populations between Tanzania and Ghana, suggesting frequent gene flow in Africa. The present study points to the importance of investigating whether biased geographical distribution for SNPs and repeat variants in the OR and SR regions affect the reactivity of human serum antibodies to variants. (C) 2011 Elsevier Ltd. All rights reserved.

Cellular and humoral immune responses against the Plasmodium vivax MSP-119 malaria vaccine candidate in individuals living in an endemic area in north-eastern Amazon region of Brazil

Abstract Background Plasmodium vivax merozoite surface protein-1 (MSP-1) is an antigen considered to be one of the leading malaria vaccine candidates. PvMSP-1 is highly immunogenic and evidences suggest that it is target for protective immunity against asexual blood stages of malaria parasites. Thus, this study aims to evaluate the acquired cellular and antibody immune responses against PvMSP-1 in individuals naturally exposed to malaria infections in a malaria-endemic area in the north-eastern Amazon region of Brazil. Methods The study was carried out in Paragominas, Pará State, in the Brazilian Amazon. Blood samples were collected from 35 individuals with uncomplicated malaria. Peripheral blood mononuclear cells were isolated and the cellular proliferation and activation was analysed in presence of 19 kDa fragment of MSP-1 (PvMSP-119) and Plasmodium falciparum PSS1 crude antigen. Antibodies IgE, IgM, IgG and IgG subclass and the levels of TNF, IFN-γ and IL-10 were measured by enzyme-linked immunosorbent assay. Results The prevalence of activated CD4+ was greater than CD8+ T cells, in both ex-vivo and in 96 h culture in presence of PvMSP-119 and PSS1 antigen. A low proliferative response against PvMSP-119 and PSS1 crude antigen after 96 h culture was observed. High plasmatic levels of IFN-γ and IL-10 as well as lower TNF levels were also detected in malaria patients. However, in the 96 h supernatant culture, the dynamics of cytokine responses differed from those depicted on plasma assays; in presence of PvMSP-119 stimulus, higher levels of TNF were noted in supernatant 96 h culture of malaria patient’s cells while low levels of IFN-γ and IL-10 were verified. High frequency of malaria patients presenting antibodies against PvMSP-119 was evidenced, regardless class or IgG subclass.PvMSP-119-induced antibodies were predominantly on non-cytophilic subclasses. Conclusions The results presented here shows that PvMSP-119 was able to induce a high cellular activation, leading to production of TNF and emphasizes the high immunogenicity of PvMSP-119 in naturally exposed individuals and, therefore, its potential as a malaria vaccine candidate.

The purine salvage enzyme hypoxanthine guanine xanthine phosphoribosyl transferase is a major target antigen for cell-mediated immunity to malaria

Although there is good evidence that immunity to the blood stages of malaria parasites can be mediated by different effector components of the adaptive immune system, target antigens for a principal component, effector CD4(+) T cells, have never been defined. We generated CD4+ T cell lines to fractions of native antigens from the blood stages of the rodent parasite, Plasmodium yoelii, and identified fraction-specific T cells that had a Th1 phenotype (producing IL-2, IFN-gamma, and tumor necrosis factor-a, but not IL-4, after antigenic stimulation). These T cells could inhibit parasite growth in recipient severe combined immunodeficient mice. N-terminal sequencing of the fraction showed identity with hypoxanthine guanine xanthine phosphoribosyl transferase (HGXPRT). Recombinant HGXPRT from the human malaria parasite, Plasmodium falciparum, activated the T cells in vitro, and immunization of normal mice with recombinant HGXPRT reduced parasite growth rates in all mice after challenge.

Immuno-potentiating activity of surfactant vesicles in DNA and subunit vaccination

Enhanced immune responses for DNA and subunit vaccines potentiated by surfactant vesicle based delivery systems outlined in the present study, provides proof of principle for the beneficial aspects of vesicle mediated vaccination. The dehydration-rehydration technique was used to entrap plasmid DNA or subunit antigens into lipid-based (liposomes) or non-ionic surfactant-based (niosomes) dehydration-rehydration vesicles (DRV). Using this procedure, it was shown that both these types of antigens can be effectively entrapped in DRV liposomes and DRV niosomes. The vesicle size of DRV niosomes was shown to be twice the diameter (~2µm) of that of their liposome counterparts. Incorporation of cryoprotectants such as sucrose in the DRV procedure resulted in reduced vesicle sizes while retaining high DNA incorporation efficiency (~95%). Transfection studies in COS 7 cells demonstrated that the choice of cationic lipid, the helper lipid, and the method of preparation, all influenced transfection efficiency indicating a strong interdependency of these factors. This phenomenon has been further reinforced when 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): cholesteryl 3b- [N-(N’ ,N’ -dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol)/DNA complexes were supplemented with non-ionic surfactants. Morphological analysis of these complexes using transmission electron microscopy and environmental scanning electron microscopy (ESEM) revealed the presence of heterogeneous structures which may be essential for an efficient transfection in addition to the fusogenic properties of DOPE. In vivo evaluation of these DNA incorporated vesicle systems in BALB/c mice showed weak antibody and cell-mediated immune (CMI) responses. Subsequent mock challenge with hepatitis B antigen demonstrated that, 1-monopalmitoyl glycerol (MP) based DRV, is a more promising DNA vaccine adjuvant. Studying these DRV systems as adjuvants for the Hepatitis B subunit antigen (HBsAg) revealed a balanced antibody/CMI response profile on the basis of the HBsAg specific antibody and cytokine responses which were higher than unadjuvated antigen. The effect of addition of MP, cholesterol and trehalose 6,6’-dibehenate (TDB) on the stability and immuno-efficacy of dimethyldioctadecylammonium bromide (DDA) vesicles was investigated. Differential scanning calorimetry showed a reduction in transition temperature of DDA vesicles by ~12°C when incorporated with surfactants. ESEM of MP based DRV system indicated an increased vesicle stability upon incorporation of antigen. Adjuvant activity of these systems tested in C57BL/6j mice against three subunit antigens i.e., mycobacterial fusion protein- Ag85B-ESAT-6, and two malarial antigens - merozoite surface protein-1, (MSP1), and glutamate rich protein, (GLURP) revealed that while MP and DDA based systems induced comparable antibody responses, DDA based systems induced powerful CMI responses.