A totally synthetic polyoxime malaria vaccine containing Plasmodium falciparum B cell and universal T cell epitopes elicits immune responses in volunteers of diverse HLA types

This open-labeled phase I study provides the first demonstration of the immunogenicity of a precisely defined synthetic polyoxime malaria vaccine in volunteers of diverse HLA types. The polyoxime, designated (T1BT(*))(4)-P3C, was constructed by chemoselective ligation, via oxime bonds, of a tetrabranched core with a peptide module containing B cell epitopes and a universal T cell epitope of the Plasmodium falciparum circumsporozoite protein. The triepitope polyoxime malaria vaccine was immunogenic in the absence of any exogenous adjuvant, using instead a core modified with the lipopeptide P3C as an endogenous adjuvant. This totally synthetic vaccine formulation can be characterized by mass spectroscopy, thus enabling the reproducible production of precisely defined vaccines for human use. The majority of the polyoxime-immunized volunteers (7/10) developed high levels of anti-repeat Abs that reacted with the native circumsporozoite on P. falciparum sporozoites. In addition, these seven volunteers all developed T cells specific for the universal epitope, termed T(*), which was originally defined using CD4(+) T cells from protected volunteers immunized with irradiated P. falciparum sporozoites. The excellent correlation of T(*)-specific cellular responses with high anti-repeat Ab titers suggests that the T(*) epitope functioned as a universal Th cell epitope, as predicted by previous peptide/HLA binding assays and by immunogenicity studies in mice of diverse H-2 haplotypes. The current phase I trial suggests that polyoximes may prove useful for the development of highly immunogenic, multicomponent synthetic vaccines for malaria, as well as for other pathogens.     

Structure of domain III of the blood-stage malaria vaccine candidate, Plasmodium falciparum apical membrane antigen 1 (AMA1)

Apical membrane antigen 1 of the malarial parasite Plasmodium falciparum (Pf AMA1) is a merozoite antigen that is considered a strong candidate for inclusion in a malaria vaccine. Antibodies reacting with disulphide bond-dependent epitopes in AMA1 block invasion of host erythrocytes by P.falciparum merozoites, and we show here that epitopes involving sites of mutations in domain III are targets of inhibitory human antibodies. The solution structure of AMA1 domain III, a 14kDa protein, has been determined using NMR spectroscopy on uniformly 15N and 13C/15N-labelled samples. The structure has a well-defined disulphide-stabilised core region separated by a disordered loop, and both the N and C-terminal regions of the molecule are unstructured. Within the disulphide-stabilised core, residues 443-447 form a turn of helix and residues 495-498 and 503-506 an anti-parallel beta-sheet with a distorted type I beta-turn centred on residues 500-501, producing a beta-hairpin-type structure. The structured region of the molecule includes all three disulphide bonds. The previously unassigned connectivities for two of these bonds could not be established with certainty from the NMR data and structure calculations, but were determined to be C490-C507 and C492-C509 from an antigenic analysis of mutated forms of this domain expressed using phage display. Naturally occurring mutations in domain III that are located far apart in the primary sequence tend to cluster in the region of the disulphide core in the three-dimensional structure of the molecule. The structure shows that nearly all the polymorphic sites have a high level of solvent accessibility, consistent with their location in epitopes recognised by protective antibodies. Even though domain III in solution contains significant regions of disorder in the structure, the disulphide-stabilised core that is structured is clearly an important element of the antigenic surface of AMA1 recognised by protective antibodies.     

Expression and purification of Plasmodium falciparum MSP-1(42): A malaria vaccine candidate

The C-terminal 42.10(3) Da portion of the merozoite surface protein (MSP-1) of the human malaria parasite Plasmodium falciparum is of interest, not only because it may constitute an essential part of a future anti-malaria vaccine, but also due to its role during the infection of erythrocytes by the parasite. We have cloned and expressed two synthetic DNA sequences encoding the two prototypic MSP-1(42) variants in E. coli. When over-produced, both proteins form insoluble aggregates which were isolated in high purity and yield. After solubilisation and refolding in vitro, both proteins were purified to homogeneity by a three-step procedure applying Ni-chelate, size exclusion and immuno-affinity chromatography. After purification, both proteins meet key criteria of preparations for clinical use. First, conformational studies suggest proper folding of the proteins, particularly in the region containing two EGF-like domains. Polyclonal serum raised against E. coli produced MSP-1(42) recognizes native MSP-1 in Plasmodium infected erythrocytes as shown by immunofluorescence.     

Differential induction of functional IgG using the Plasmodium falciparum placental malaria vaccine candidate VAR2CSA

Background

In Plasmodium falciparum malaria endemic areas placental malaria (PM) is an important complication of malaria. The recurrence of malaria in primigravidae women irrespective of acquired protection during childhood is caused by the interaction between the parasite-expressed VAR2CSA antigen and chondroitin sulfate A (CSA) in the placental intervillous space and lack of protective antibodies. PM impairs fetal development mainly by excessive inflammation processes. After infections during pregnancy women acquire immunity to PM conferred by antibodies against VAR2CSA. Ideally, a vaccine against PM will induce antibody-mediated immune responses that block the adhesion of infected erythrocytes (IE) in the placenta.

Principal Findings

We have previously shown that antibodies raised in rat against individual domains of VAR2CSA can block IE binding to CSA. In this study we have immunized mice, rats and rabbits with each individual domain and the full-length protein corresponding to the FCR3 VAR2CSA variant. We found there is an inherently higher immunogenicity of C-terminal domains compared to N-terminally located domains. This was irrespective of whether antibodies were induced against single domains or the full-length protein. Species-specific antibody responses were also found, these were mainly directed against single domains and not the full-length VAR2CSA protein.

Conclusions/Significance

Binding inhibitory antibodies appeared to be against conformational B-cell epitopes. Non-binding inhibitory antibodies reacted highly against the C-terminal end of the VAR2CSA molecule especially the highly polymorphic DBL6ε domain. Differential species-specific induction of antibody responses may allow for more direct analysis of functional versus non-functional B-cell epitopes.     

The role of Plasmodium falciparum var genes in malaria in pregnancy

Sequestration of Plasmodium falciparum-infected erythrocytes in the placenta is responsible for many of the harmful effects of malaria during pregnancy. Sequestration occurs as a result of parasite adhesion molecules expressed on the surface of infected erythrocytes binding to host receptors in the placenta such as chondroitin sulphate A (CSA). Identification of the parasite ligand(s) responsible for placental adhesion could lead to the development of a vaccine to induce antibodies to prevent placental sequestration. Such a vaccine would reduce the maternal anaemia and infant deaths that are associated with malaria in pregnancy. Current research indicates that the parasite ligands mediating placental adhesion may be members of the P. falciparum variant surface antigen family PfEMP1, encoded by var genes. Two relatively well-conserved subfamilies of var genes have been implicated in placental adhesion, however, their role remains controversial. This review examines the evidence for and against the involvement of var genes in placental adhesion, and considers whether the most appropriate vaccine candidates have yet been identified.     

Plasmodium falciparum: immunogenicity of alum-adsorbed clinical-grade TBV25-28, a yeast-secreted malaria transmission-blocking vaccine candidate

Gozar, M. M. G., Muratova, O., Keister, D. B., Kensil, C. R., Price, V. L., and Kaslow, D. C. 2001. Plasmodium falciparum: Immunogenicity of alum-adsorbed clinical-grade TBV25-28, a yeast-secreted malaria transmission-blocking vaccine candidate. Experimental Parasitology 97, 61-69. The fusion of Pfs25 and Pfs28, two major surface antigens on zygotes and ookinetes of Plasmodium falciparum, as a single recombinant protein (TBV25-28) was previously shown to elicit potent transmission-blocking antibodies in mice. Clinical-grade TBV25-28 was subsequently manufactured and its potency was evaluated in rabbits. Rabbits received three doses of either clinical-grade TBV25H or clinical-grade TBV25-28 adsorbed to alum with or without QS-21. As measured in a standard membrane-feeding assay, addition of QS-21 to the formulations appeared to enhance transmission-blocking potency of rabbit sera after two vaccinations but not after three vaccinations. Surprisingly, TBV25H elicited more potent transmission-blocking antibodies than did TBV25-28, a result strikingly different from those of previous mouse experiments using research-grade TBV25-28. The apparent decrease in potency of clinical-grade TBV25-28 in rabbits appears to reflect an enhancement in potency of clinical-grade TBV25H in a new formulation rather than simply a species difference in immunogenicity of TBV25-28.     

Unique T cell effector functions elicited by Plasmodium falciparum epitopes in malaria-exposed Africans tested by three T cell assays

Natural immunity to malaria is characterized by low level CD4 T cell reactivity detected by either lymphoproliferation or IFN-gamma secretion. Here we show a doubling in the detection rate of responders to the carboxyl terminus of circumsporozoite protein (CS) of Plasmodium falciparum by employing three T cell assays simultaneously: rapid IFN-gamma secretion (ex vivo ELISPOT), IFN-gamma secretion after reactivation of memory T cells and expansion in vitro (cultured ELISPOT), and lymphoproliferation. Remarkably, for no individual peptide did a positive response for one T cell effector function correlate with any other. Thus these CS epitopes elicited unique T cell response patterns in malaria-exposed donors. Novel or important epitope responses may therefore be missed if only one T cell assay is employed. A borderline correlation was found between anti-CS Ab levels and proliferative responses, but no correlation was found with ex vivo or cultured IFN-gamma responses. This suggested that the proliferating population, but not the IFN-gamma-secreting cells, contained cells that provide help for Ab production. The data suggest that natural immunity to malaria is a complex function of T cell subgroups with different effector functions and has important implications for future studies of natural T cell immunity.     

Plasmodium falciparum: restricted polymorphism of T cell epitopes of the circumsporozoite protein in Brazil.

We examined the extent of variation of the 3' region of the circumsporozoite gene among Plasmodium falciparum isolates through amplification of a selected DNA fragment followed by DNA sequencing. A total of 32 isolates were analyzed, of which 24 were from Amazon endemic areas in Brazil and 8 from widely separated geographical regions in the world. Among Brazilian isolates only 2 variants were detected: 19 displayed the same sequence of strain 7G8 whereas the 4 remaining isolates differed from the 7G8 strain at five nucleotide positions which also led to amino acid changes. Variation was restricted to one of the T-helper epitopes while the sequence identified as a cytotoxic T cell epitope was conserved in all Brazilian isolates. P. falciparum samples from other geographical regions in the world showed sequences distinct from those of Brazilian isolates. However, some constancy could be observed within that variation. For instance, the most frequent nucleotide substitutions, from A and C at nucleotide positions 1015 and 1024, were the same in all isolates.     

Global diversity and balancing selection of 23 leading Plasmodium falciparum candidate vaccine antigens

Investigation of the diversity of malaria parasite antigens can help prioritize and validate them as vaccine candidates and identify the most common variants for inclusion in vaccine formulations. Studies of vaccine candidates of the most virulent human malaria parasite, Plasmodium falciparum, have focused on a handful of well-known antigens, while several others have never been studied. Here we examine the global diversity and population structure of leading vaccine candidate antigens of P. falciparum using the MalariaGEN Pf3K (version 5.1) resource, comprising more than 2600 genomes from 15 malaria endemic countries. A stringent variant calling pipeline was used to extract high quality antigen gene ‘haplotypes’ from the global dataset and a new R-package named VaxPack was used to streamline population genetic analyses. In addition, a newly developed algorithm that enables spatial averaging of selection pressure on 3D protein structures was applied to the dataset. We analysed the genes encoding 23 leading and novel candidate malaria vaccine antigens including csp, trap, eba175, ama1, rh5, and CelTOS. Our analysis shows that current malaria vaccine formulations are based on rare haplotypes and thus may have limited efficacy against natural parasite populations. High levels of diversity with evidence of balancing selection was detected for most of the erythrocytic and pre-erythrocytic antigens. Measures of natural selection were then mapped to 3D protein structures to predict targets of functional antibodies. For some antigens, geographical variation in the intensity and distribution of these signals on the 3D structure suggests adaptation to different human host or mosquito vector populations. This study provides an essential framework for the diversity of P. falciparum antigens to be considered in the design of the next generation of malaria vaccines.     

Diversity in two candidate malaria vaccine antigens in different Indian strains ofPlasmodium falciparum

Polymorphism in two malarial antigens, merozoite surface antigen-1 (MSA-1) and ring erythrocyte surface antigen (RESA), has been characterized in four different Indian strains ofPlasmodium falciparum. The Indian strains were obtained from two malaria endemic regions of India, viz. Surat (Gujarat) and Delhi, and established in culture. Monoclonal and polyclonal antibodies raised against different domains of these antigens were used in the study. In MSA-1 a novel intragenic crossover was detected in the central conserved domain in two of the Indian strains. The repeat domain of RESA was found to be absent in the two strains ofP. falciparum isolated from Surat. These differences in immunoreactivity have been extended to the DNA level by appropriate PCR studies. MSA-1 and RESA are candidate vaccine antigens and these diversities will have an important bearing on the design of a suitable malaria vaccine.     

Plasmodium falciparum: worldwide sequence diversity and evolution of the malaria vaccine candidate merozoite surface protein-2 (MSP-2)

We examined patterns and putative mechanisms of sequence diversification in the merozoite surface protein-2 (MSP-2) of Plasmodium falciparum, a major dimorphic malaria vaccine candidate antigen, by analyzing 448 msp-2 alleles from all continents. We describe several nucleotide replacements, insertion and deletion events, frameshift mutations, and proliferations of repeat units that generate the extraordinary diversity found in msp-2 alleles. We discuss the role of positive selection exerted by naturally acquired type- and variant-specific immunity in maintaining the observed levels of polymorphism and suggest that this is the most likely explanation for the significant excess of nonsynonymous nucleotide replacements found in dimorphic msp-2 domains. Hybrid sequences created by meiotic recombination between alleles of different dimorphic types were observed in few (3.1%) isolates, mostly from Africa. We found no evidence for an extremely ancient origin of allelic dimorphism at the msp-2 locus, predating P. falciparum speciation, in contrast with recent findings for other surface malarial antigens.     

Nonpolymorphic regions of p190, a protein of the Plasmodium falciparum erythrocytic stage, contain both T and B cell epitopes

Two conserved regions from the genetically polymorphic p190 molecule of the malaria parasite Plasmodium falciparum have previously been expressed in Escherichia coli as separate polypeptides (190.L and 190.M) or as a single fusion protein (190.N). In the present study we investigated whether human B and T lymphocytes recognize these conserved regions. The more amino-terminal region, 190.L (corresponding to residues 188-363 of the encoded protein sequence) reacted preferentially with sera from donors living in a malaria-endemic area. Also, EBV-transformed B cells, from a healthy donor living in a malaria-mesoendemic area, were fused with a human-mouse hybrid line (SPM4-0), yielding two hybridomas whose products recognized both 190.L and the fusion protein 190.N, but not the 190.M polypeptide. A large number of p190-specific T cell clones were obtained from PBMC of a noninfected donor, after in vitro stimulation with the recombinant fusion protein 190.N. The clones reacted with intact, parasite-derived p190, as well as either 190.L or 190.M. Four clones that recognized the more amino-terminal fragment also responded to infected E. According to these results the more amino-terminal conserved sequences of p190 have the requisites to be immunogenic in humans.     

The MYST family histone acetyltransferase regulates gene expression and cell cycle in malaria parasite Plasmodium falciparum

Histone lysine acetylation, normally associated with euchromatin and active genes, is regulated by different families of histone acetyltransferases (HATs). A single Plasmodium falciparum MYST (PfMYST) HAT was expressed as a long and a short version in intraerythrocytic stages. Whereas the recombinant PfMYST expressed in prokaryotes and insect cells did not show HAT activity, recombinant PfMYST purified from the parasites exhibited a predilection to acetylate histone H4 in vitro at K5, K8, K12 and K16. Tagging PfMYST with the green fluorescent protein at the C‐terminus showed that PfMYST protein was localized in both the nucleus and cytoplasm. Consistent with the importance of H4 acetylation in var gene expression, PfMYST was recruited to the active var promoter. Attempts to disrupt PfMYST were not successful, suggesting that PfMYST is essential for asexual intraerythrocytic growth. However, overexpression of the long, active or a truncated, non‐active version of PfMYST by stable integration of the expression cassette in the parasite genome resulted in changes of H4 acetylation and cell cycle progression. Furthermore, parasites with PfMYST overexpression showed changes in sensitivity to DNA‐damaging agents. Collectively, this study showed that PfMYST plays important roles in cellular processes such as gene activation, cell cycle control and DNA repair.