Antimalarial Vaccine Development Research Articles

Malaria vaccine development

According to the statistics of the World Health Organization, malaria is still one of the main diseases affecting human health, especially in Africa, and inflicts a heavy disease burden and a huge economic burden in endemic countries. At present, the widespread emergence of antimalarial drug resistance and unresolved drug availability issues have led researchers to turn their attention to the development of antimalarial vaccines. This review aims at highlighting the recent development of malaria vaccines and discussing the challenges.

The molecular basis for peptide-based antimalarial vaccine development targeting erythrocyte invasion by P. falciparum

This work describes a methodology for developing a minimal, subunit-based, multi-epitope, multi-stage, chemically-synthesised, anti-Plasmodium falciparum malaria vaccine. Some modified high activity binding peptides (mHABPs) derived from functionally relevant P. falciparum MSP, RH5 and AMA-1 conserved amino acid regions (cHABPs) for parasite binding to and invasion of red blood cells (RBC) were selected. They were highly immunogenic as assessed by indirect immunofluorescence (IFA) and Western blot (WB) assays and protective immune response-inducers against malarial challenge in the Aotus monkey experimental model. NetMHCIIpan 4.0 was used for predicting peptide-Aotus/human major histocompatibility class II (MHCII) binding affinity in silico due to the similarity between Aotus and human immune system molecules; ∼50% of Aotus MHCII allele molecules have a counterpart in the human immune system, being Aotus-specific, whilst others enabled recognition of their human counterparts. Some peptides’ 1H-NMR-assessed structural conformation was determined to explain residue modifications in mHABPs inducing secondary structure changes. These directly influenced immunological behaviour, thereby highlighting the relationship with MHCII antigen presentation. The data obtained in such functional, immunological, structural and predictive approach suggested that some of these peptides could be excellent components of a fully-protective antimalarial vaccine.

Transient Expression of Plasmodium berghei MSP8 and HAP2 in the Marine Protozoan Parasite Perkinsus marinus.

Perkinsus marinus is a protozoan parasite of molluscs that can be propagated in vitro in a defined culture medium, in the absence of host cells. We previously reported that P. marinus trophozoites can be transfected with high efficiency by electroporation using a plasmid based on MOE, a highly expressed gene, and proposed its potential use as a "pseudoparasite." This is a novel gene expression platform for parasites of medical relevance for which the choice of the surrogate organism is based on phylogenetic affinity to the parasite of interest, while taking advantage of the whole engineered surrogate organism as a vaccination adjuvant. Here we improved the original transfection plasmid by incorporating a multicloning site, an enterokinase recognition sequence upstream of GFP, and a His-tag and demonstrate its potential suitability for the heterologous expression of Plasmodium sp. genes relevant to the development of anti-malarial vaccines. Plasmodium berghei HAP2 and MSP8, currently considered candidate genes for a malaria vaccine, were cloned into p[MOE]:GFP, and the constructs were used to transfect P. marinus trophozoites. Within 48 hr of transfection we observed fluorescent cells indicating that the P. berghei genes fused to GFP were expressed. The expression appeared to be transient for both P. berghei genes, as florescence of the transfectants diminished gradually over time. Although this heterologous expression system will require optimization for integration and constitutive expression of Plasmodium genes, our results represent attainment of proof for the "pseudoparasite" concept we previously proposed, as we show that the engineered P. marinus system has the potential to become a surrogate system suitable for expression of Plasmodium spp. genes of interest, which could eventually be used as a malaria vaccine delivery platform. The aim of the present study was to test the ability of marine protozoan parasite P. marinus to express genes of P. berghei .

Effect of clodronate-liposome injections on Plasmodium falciparum infection in neo-tropical primates Saimiri sciureus

Background One of the constraints in the development of antimalarial vaccines is the limited availability of experimental models that reliably reproduce the infection and the immunopathology observed in human malaria. The best experimental models are Saimiri and Aotus neotropical primates, but they have as important limitations, the need for splenectomy for the expression of high and consistent parasitemias. The clodronate-liposome (CL) has been used to deplete macrophages in several experimental models and we tested the hypothesis that their use in Saimiri would allow the development of consistent P. falciparum parasitemias without the need for splenectomy.

Gauche+ side-chain orientation as a key factor in the search for an immunogenic peptide mixture leading to a complete fully protective vaccine

Topological and stereo-electron characteristics are essential in major histocompability class II–peptide–T–cell receptor (MHC–p–TCR) complex formation for inducing an appropriate immune response. Modified high activity binding peptides (mHABPs) were synthesised for complete full protection antimalarial vaccine development producing a large panel of individually fully protection-inducing protein structures (FPIPS) and very high long-lasting antibody-inducing (VHLLAI) mHABPs. Most of those which did not interfere, compete, inhibit or suppress their individual VHLLAI or FPIPS activity contained or displayed a polyproline II-like (PPIIL) structure when mixed. Here we show that amino acid side-chains located in peptide binding region (PBR) positions p3 and p7 displayed specific electron charges and side-chain gauche+ orientation for interacting with the TCR. Based on the above, and previously described physicochemical principles, non-interfering, long-lasting, full protection-inducing, multi-epitope, multistage, minimal subunit-based chemically synthesised mHABP mixtures can be designed for developing vaccines against diseases scourging humankind, malaria being one of them.

Reduced protective effect of Plasmodium berghei immunization by concurrent Schistosoma mansoni infection

Studies on concomitant schistosomiasis and human and experimental malaria have shown a variation in the immunospecific response, as well as an increase in the severity of both parasitoses. In the present study, a murine co-infection model was used to determine the effects of a co-infection with Schistosoma mansoni and Plasmodium berghei on the protective immunity acquired by repeated malarial infections and subsequent curative treatment with chloroquine. Our results have demonstrated that, compared to an infection with P. berghei only, the co-infection increases the malarial parasitaemia and decreases the survival rate. Indeed, mice that were immunized by infection and treatment with drug displayed no mortality whereas co-infected mice showed a reduced protective efficacy of immunization against P. berghei (mortality > 60%). Interestingly, this high mortality rate was not associated with high levels of parasitaemia. Our findings support the idea of a suppressive effect of a Schistosoma co-infection on the anti-malarial protection by immunization. This result reveals a possible drawback of the development of anti-malarial vaccines, especially considering the wide endemic areas for both parasitoses.

Polymorphism study of rhoptry associated membrane antigen (RAMA) gene of Plasmodium falciparum—A putative vaccine candidate

The antigenic diversity of Plasmodium falciparum is one of the major obstacles to antimalarial vaccine development. Thus, it becomes obvious to search for a protein that is fairly conserved and is necessary for the parasite to survive in the host cell. Rhoptry associated membrane antigen (RAMA) plays a potential role in parasite biology and invasion. The C-terminal end of RAMA is shown to have protective immune responses and binds to the erythrocyte membrane. In this work, we have studied the polymorphism of RAMA gene in the C-terminal end from 1525 to 3196 nucleotide (nt) in 230 samples. The presence of few variants suggests RAMA to be under a balancing selection. The above criterion of restricted antigenic diversity and a protective immune response towards the C-terminal end makes RAMA a strong vaccine candidate.

A pre-PEXEL histidine-rich protein II erythrocyte binding peptide as a new way for anti-malarial vaccine development

The Plasmodium falciparum malaria parasite produces several proteins characterised by an unusually high histidine content in infected red blood cells (iRBC). The histidine-rich protein II (HRP-II) is synthesised throughout the parasite’s asexual and gametocyte stages, transported through the parasitophorous vacuole (PV) to iRBC cytosol and membrane and released to the bloodstream via a PEXEL motif. Immunogenicity and protection-inducing studies were begun with an RBC high activity binding peptide (HABP) from this protein named 6800 (preceding the PEXEL motif) in the experimental Aotus monkey model. Modifying critical residues (determined by glycine scanning in this HABP) induced immunogenicity and protection against experimental challenge. Native 6800 did not bind to any HLA-DRβ 1 ∗ molecule, but these modified HABPs acquired the ability to specifically bind to HLA-DRβ 1 ∗0701. 1H NMR studies revealed that whilst 6800 had a random structure, modified immunogenic and protection-inducing 24230 displayed very short α-helical segments allowing appropriate binding to the MHCII-pep-TCR complex. Modifications in conserved HABPs preceding PEXEL motifs thus open up new avenues for subunit-based, multi-component synthetic anti-malarial vaccine development.

Functional, structural, and immunological compartmentalisation of malaria invasive proteins

Conserved Plasmodium falciparum merozoite high activity binding peptides (HABPs) involved in red blood cell (RBC) invasion which are present in merozoite surface proteins (MSPs) involved in attachment, rolling over RBC, those derived from soluble proteins loosely bound to the membrane, and those present in microneme and rhoptry organelles have an α-helical structure and bind with high affinity to HLA-DR52 molecules. On the contrary, conserved HABPs belonging to molecules anchored to the membrane by a GPI tail, or a transmembranal region, or those molecules presenting PEXEL motifs have a strand, turn or unordered configuration and bind with high affinity to HLA-DR53 molecules. Such functional, cellular, structural, and immunological compartmentalisation has tremendous implications in subunit-based, multi-epitope, synthetic, anti-malarial vaccine development.

Comparative molecular and three-dimensional analysis of the peptide–MHC II binding region in both human and Aotus MHC-DRB molecules confirms their usefulness in antimalarial vaccine development

A vaccine against malaria is desperately needed, and Aotus monkeys are highly susceptible to experimental infection with malarial parasites. A thorough analysis of this monkey's immune system molecules was thus undertaken in our institute. Cloning and sequencing, followed by three-dimensional analysis, has revealed high homology with some HLA-DRB1 molecules in terms of their peptide binding region pockets. Molecules such as HLA-DRB1*03, 11, 08, and HLA-DRB1*04 are so similar to Aotus MHC-DRB molecules that peptides identified as binding to these molecules and inducing protective immunity in these monkeys could be used in humans without further refinement, while small modifications seem to be needed for those binding to HLA-DRB1*07, HLA-DRB1*15, 16, and HLA-DRB1*10-like molecules, making this New World monkey an excellent model for tailor-made vaccine development, especially against malaria.

Polymorphism in two merozoite surface proteins of Plasmodium falciparum isolates from Gabon

BackgroundPlasmodium falciparum antigenic diversity and polymorphism confuses the issue of antimalarial vaccine development. Merozoite surface protein (MSP)-1 and -2 are two highly polymorphic vaccine candidates. Characterisation of their precise polymorphism in endemic regions may facilitate the design of an effective vaccine.MethodsIsolates obtained in 52 Gabonese children presenting with uncomplicated malaria were genotyped by nested-PCR of msp-1 block 2, and msp-2 block 3, to analyze both parasite population polymorphism and clone fluctuations.ResultsTwenty-five and 19 different alleles were respectively obtained for msp-1 and msp-2 loci, the RO33 family of msp-1 being poorly polymorphic. Four cases of non-random distribution of alleles were reported of the FC27, and/or 3D7 families of msp-2. All but two isolates were composed of more than one genotype, and the multiplicity of infection (MOI) was 4.0. Neither parasite density nor age was related to MOI. Clone fluctuations were studied for ten subjects who were sampled again at reappearance of parasites in blood. Disappearance and reappearance of alleles were observed following treatment, suggesting difficulties in assessing polymorphism and in distinguishing reinfection from recrudescence.ConclusionP. falciparum polymorphism is extensive in Southeast Gabon, and most of infections are composed of multiple clones. The fluctuation of clones contributes to parasite diversity.

Mosquito immune responses and malaria transmission: lessons from insect model systems and implications for vertebrate innate immunity and vaccine development

The introduction of novel biochemical, genetic, molecular and cell biology tools to the study of insect immunity has generated an information explosion in recent years. Due to the biodiversity of insects, complementary model systems have been developed. The conceptual framework built based on these systems is used to discuss our current understanding of mosquito immune responses and their implications for malaria transmission. The areas of insect and vertebrate innate immunity are merging as new information confirms the remarkable extent of the evolutionary conservation, at a molecular level, in the signaling pathways mediating these responses in such distant species. Our current understanding of the molecular language that allows the vertebrate innate immune system to identify parasites, such as malaria, and direct the acquired immune system to mount a protective immune response is very limited. Insect vectors of parasitic diseases, such as mosquitoes, could represent excellent models to understand the molecular responses of epithelial cells to parasite invasion. This information could broaden our understanding of vertebrate responses to parasitic infection and could have extensive implications for anti-malarial vaccine development.

The Crystal Structure of C-Terminal Merozoite Surface Protein 1 at 1.8 Å Resolution, a Highly Protective Malaria Vaccine Candidate

The C-terminal proteolytic processing product of merozoite surface protein 1 (MSP1) appears essential for successful erythrocyte invasion by the malarial parasite, Plasmodium. We have determined the crystal structure at 1.8 Å resolution of a soluble baculovirus-recombinant form of the protein from P. cynomolgi, which confers excellent protective efficacy in primate vaccination trials. The structure comprises two EGF-like domains, and sequence comparisons strongly suggest that the same conformation is present in all species of Plasmodium, including P. falciparum and P. vivax, which are pathogenic in man. In particular, conserved interdomain contacts between the two EGF modules should preserve the compact form of the molecule in all species. Implications of the crystal structure for anti-malarial vaccine development are discussed.

Localization of HLA-A2.1-restricted T cell epitopes in the circumsporozoite protein of Plasmodium falciparum.

Localization of human MHC class I-restricted T cell epitopes in the circumsporozoite (CS) protein of the human parasite Plasmodium falciparum is an important objective in the development of antimalarial vaccines. To this purpose, we synthesized a series of overlapping synthetic 20-mer peptides, spanning the entire sequence of the 7G8 CS molecule except for the central repeat B cell domain. The P.f.CS peptides were first tested for their ability to bind to the human MHC class I HLA-A2.1 molecule on T2, a human cell line. Subsequently, the use of a series of shorter peptide analogues allowed us to determine the optimal A2.1 binding sequence present in several of the 20-mers. Binding P.f.CS peptides were further tested for their capacity to activate PBL from HLA-A2.1+ immune donors living in a malaria-endemic area. Specific IFN-gamma production was detected in the supernatant of cultures of PBL from exposed individuals. Cytotoxic T cell lines and clones were derived from the PBL of one responder, and their activity was shown to be HLA-A2.1-restricted and specific for the peptide 334-342 of the CS protein. In addition, double transgenic HLA-A2.1 x human beta 2-microglobulin mice were immunized with peptide 1-10 of the CS protein. T cells derived from immune lymph nodes displayed a peptide-specific HLA-A2.1-restricted cytolytic activity after one in vitro stimulation.