Preparation and characterization of poly(lactic-co-glycolic acid) microparticles containing DNA molecules encoding a malaria vaccine candidate

Background A novel ultrasonic atomization approach for the formulation of biodegradable poly(lactic-co-glycolic acid) (PLGA) microparticles of a malaria DNA vaccine is presented. A 40 kHz ultrasonic atomization device was used to create the microparticles from a feedstock containing 5 volumes of 0.5% w/v PLGA in acetone and 1 volume of condensed DNA which was fed at a flow rate of 18ml h-1. The plasmid DNA vectors encoding a malaria protein were condensed with a cationic polymer before atomization. Results High levels of gene expression in vitro were observed in COS-7 cells transfected with condensed DNA at a nitrogen to phosphate (N/P) ratio of 10. At this N/P ratio, the condensed DNA exhibited a monodispersed nanoparticle size (Z-average diameter of 60.8 nm) and a highly positive zeta potential of 38.8mV. The microparticle formulations of malaria DNA vaccine were quality assessed and it was shown that themicroparticles displayed high encapsulation efficiencies between 82-96% and a narrow size distribution in the range of 0.8-1.9 μm. In vitro release profile revealed that approximately 82% of the DNA was released within 30 days via a predominantly diffusion controlledmass transfer system. Conclusions This ultrasonic atomization technique showed excellent particle size reproducibility and displayed potential as an industrially viable approach for the formulation of controlled release particles.

Rapid production of a plasmid DNA encoding a malaria vaccine candidate via amino-functionalized poly(GMA-co-EDMA) monolith

Malaria is a global health problem; an effective vaccine is urgently needed. Due to the relative poverty and lack of infrastructure in malaria endemic areas, DNA-based vaccines that are stable at ambient temperatures and easy to formulate have great potential. While attention has been focused mainly on antigen selection, vector design and efficacy assessment, the development of a rapid and commercially viable process to manufacture DNA is generally overlooked. We report here a continuous purification technique employing an optimized stationary adsorbent to allow high-vaccine recovery, low-processing time, and, hence, high-productivity. A 40.0 mL monolithic stationary phase was synthesized and functionalized with amino groups from 2-Chloro-N,N- diethylethylamine hydrochloride for anion-exchange isolation of a plasmid DNA (pDNA) that encodes a malaria vaccine candidate, VR1020-PyMSP4/5. Physical characterization of the monolithic polymer showed a macroporous material with a modal pore diameter of 750 nm. The final vaccine product isolated after 3 min elution was homogeneous supercoiled plasmid with gDNA, RNA and protein levels in keeping with clinical regulatory standards. Toxicological studies of the pVR1020-PyMSP4/5 showed a minimum endotoxin level of 0.28 EU/m.g pDNA. This cost-effective technique is cGMP compatible and highly scalable for the production of DNA-based vaccines in commercial quantities, when such vaccines prove to be effective against malaria. © 2008 American Institute of Chemical Engineers.

Malaria Vaccine Adjuvants: Latest Update and Challenges in Preclinical and Clinical Research

There is no malaria vaccine currently available, and the most advanced candidate has recently reported a modest 30% efficacy against clinical malaria. Although many efforts have been dedicated to achieve this goal, the research was mainly directed to identify antigenic targets. Nevertheless, the latest progresses on understanding how immune system works and the data recovered from vaccination studies have conferred to the vaccine formulation its deserved relevance. Additionally to the antigen nature, the manner in which it is presented (delivery adjuvants) as well as the immunostimulatory effect of the formulation components (immunostimulants) modulates the immune response elicited. Protective immunity against malaria requires the induction of humoral, antibody-dependent cellular inhibition (ADCI) and effector and memory cell responses. This review summarizes the status of adjuvants that have been or are being employed in the malaria vaccine development, focusing on the pharmaceutical and immunological aspects, as well as on their immunization outcomings at clinical and preclinical stages.

Malaria vaccine candidate: design of a multivalent subunit α-helical coiled coil poly-epitope.

A new strategy for the rapid identification of new malaria antigens based on protein structural motifs was previously described. We identified and evaluated the malaria vaccine potential of fragments of several malaria antigens containing α-helical coiled coil protein motifs. By taking advantage of the relatively short size of these structural fragments, we constructed different poly-epitopes in which 3 or 4 of these segments were joined together via a non-immunogenic linker. Only peptides that are targets of human antibodies with anti-parasite in vitro biological activities were incorporated. One of the constructs, P181, was well recognized by sera and peripheral blood mononuclear cells (PBMC) of adults living in malaria-endemic areas. Affinity purified antigen-specific human antibodies and sera from P181-immunized mice recognised native proteins on malaria-infected erythrocytes in both immunofluorescence and western blot assays. In addition, specific antibodies inhibited parasite development in an antibody dependent cellular inhibition (ADCI) assay. Naturally induced antigen-specific human antibodies were at high titers and associated with clinical protection from malaria in longitudinal follow-up studies in Senegal.

New malaria vaccine candidates based on the Plasmodium vivax Merozoite Surface Protein-1 and the TLR-5 agonist Salmonella Typhimurium FliC flagellin

The present study evaluated the immunogenicity of new malaria vaccine formulations based on the 19 kDa C-terminal fragment of Plasmodium vivax Merozoite Surface Protein-1 (MSP1(19)) and the Salmonella enterica serovar Typhimurium flagellin (FIiC), a Toll-like receptor 5 (TLR5) agonist. FHC was used as an adjuvant either admixed or genetically linked to the P. vivax MSP1(19) and administered to C57BL/6 mice via parenteral (s.c.) or mucosal (i.n.) routes. The recombinant fusion protein preserved MSP1(19) epitopes recognized by Sera collected from P. vivax infected humans and TLR5 agonist activity. Mice parenterally immunized with recombinant P vivax MSPI 19 in the presence of FliC, either admixed or genetically linked, elicited strong and long-lasting MSP1 (19)-specific systemic antibody responses with a prevailing IgG1 subclass response. Incorporation of another TLR agonist, CpG ODN 1826, resulted in a more balanced response, as evaluated by the IgG1/IgG2c ratio, and higher cell-mediated immune response measured by interferon-gamma secretion. Finally, we show that MSPI 19-specific antibodies recognized the native protein expressed on the surface of P. vivax parasites harvested from infected humans. The present report proposes a new class of malaria vaccine formulation based on the use of malaria antigens and the innate immunity agonist FliC. it contains intrinsic adjuvant properties and enhanced ability to induce specific humoral and cellular immune responses when administered alone or in combination with other adjuvants. (C) 2008 Elsevier Ltd. All rights reserved.

Evolutionary dynamics of the immunodominant repeats of the Plasmodium vivax malaria-vaccine candidate circumsporozoite protein (CSP)

The circumsporozoite protein (CSP) of Plasmodium vivax, a major target for malaria vaccine development, has immunodominant B-cell epitopes mapped to central nonapeptide repeat arrays. To determine whether rearrangements of repeat motifs during mitotic DNA replication of parasites create significant CSP diversity under conditions of low effective meiotic recombination rates, we examined csp alleles from sympatric P. vivax isolates systematically sampled from an area of low malaria endemicity in Brazil over a period of 14 months. Nine unique csp types, comprising six different nona peptide repeats, were observed in 45 isolates analyzed. Identical or nearly identical repeats predominated in most arrays, consistent with their recent expansion. We found strong linkage disequilibrium at sites across the chromosome 8 segment flanking the csp locus, consistent with rare meiotic recombination in this region. We conclude that CSP repeat diversity may not be severely constrained by rare meiotic recombination in areas of low malaria endemicity. New repeat variants may be readily created by nonhomologous recombination even when meiotic recombination is rare, with potential implications for CSP-based vaccine development. (C) 2010 Elsevier B.V. All rights reserved.

Functional analysis of the leading malaria vaccine candidate AMA-1 reveals an essential role for the cytoplasmic domain in the invasion process

A key process in the lifecycle of the malaria parasite Plasmodium falciparum is the fast invasion of human erythrocytes. Entry into the host cell requires the apical membrane antigen 1 (AMA-1), a type I transmembrane protein located in the micronemes of the merozoite. Although AMA-1 is evolving into the leading blood-stage malaria vaccine candidate, its precise role in invasion is still unclear. We investigate AMA-1 function using live video microscopy in the absence and presence of an AMA-1 inhibitory peptide. This data reveals a crucial function of AMA-1 during the primary contact period upstream of the entry process at around the time of moving junction formation. We generate a Plasmodium falciparum cell line that expresses a functional GFP-tagged AMA-1. This allows the visualization of the dynamics of AMA-1 in live parasites. We functionally validate the ectopically expressed AMA-1 by establishing a complementation assay based on strain-specific inhibition. This method provides the basis for the functional analysis of essential genes that are refractory to any genetic manipulation. Using the complementation assay, we show that the cytoplasmic domain of AMA-1 is not required for correct trafficking and surface translocation but is essential for AMA-1 function. Although this function can be mimicked by the highly conserved cytoplasmic domains of P. vivax and P. berghei, the exchange with the heterologous domain of the microneme protein EBA-175 or the rhoptry protein Rh2b leads to a loss of function. We identify several residues in the cytoplasmic tail that are essential for AMA-1 function. We validate this data using additional transgenic parasite lines expressing AMA-1 mutants with TY1 epitopes. We show that the cytoplasmic domain of AMA-1 is phosphorylated. Mutational analysis suggests an important role for the phosphorylation in the invasion process, which might translate into novel therapeutic strategies.

Origins of sequence diversity in the malaria vaccine candidate merozoite surface protein-2 (MSP-2) in Amazonian isolates of Plasmodium falciparum

The recent evolution of Plasmodium falciparum is at odds with the extensive polymorphism found in most genes coding for antigens. Here, we examined the patterns and putative mechanisms of sequence diversification in the merozoite surface protein-2 (MSP-2), a major malarial repetitive surface antigen. We compared the msp-2 gene sequences from closely related clones derived from sympatric parasite isolates from Brazilian Amazonia and used microsatellite typing to examine, in these same clones, the haplotype background of chromosome 2, where msp-2 is located. We found examples of msp-2 sequence rearrangements putatively created by nonreciprocal recombinational events, such as replication slippage and gene conversion, while maintaining the chromosome haplotype. We conclude that these nonreciprocal recombination events may represent a major source of antigenic diversity in MSP-2 in P falciparum populations with low rates of classical meiotic recombination. (c) 2006 Elsevier B.V. All rights reserved.

Malaria vaccine: roadblocks and possible solutions

Malaria remains the most prevalent and devastating parasitic disease worldwide. Vaccination is considered to be an approach that will complement other strategies for prevention and control of the disease in the future. In the last 10 years, intense studies aimed at the development of a malaria vaccine have provided important knowledge of the nature of the host immunological mechanisms of protection and their respective target antigens. It became well established that protective immune responses can be generated against the distinct stages of Plasmodium. However, in general, protective immune responses are directed at stage-specific antigens. The elucidation of the primary structure of these antigens made possible the generation of synthetic and recombinant proteins that are being extensively used in experimental immunizations against the infection. Today, several epitopes of limited polymorphism have been described and protective immunity can be generated by immunization with them. These epitopes are being tested as primary candidates for a subunit vaccine against malaria. Here we critically review the major roadblocks for the development of a malaria vaccine and provide some insight on how these problems are being solved.

Humoral immune responses against the malaria vaccine candidate antigen Plasmodium vivax AMA-1 and IL-4 gene polymorphisms in individuals living in an endemic area of the Brazilian Amazon

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

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.

Naturally acquired antibodies to Plasmodium vivax blood-stage vaccine candidates (PvMSP-1(19) and PvMSP-3 alpha(359-798)) and their relationship with hematological features in malaria patients from the Brazilian Amazon

An important step when designing a vaccine is identifying the antigens that function as targets of naturally acquired antibodies. We investigated specific antibody responses against two Plasmodium vivax vaccine candidates, PvMSP-1(19) and PvMSP-3 alpha(359-798). Moreover, we assessed the relationship between these antibodies and morbidity parameters. PvMSP-1(19) was the most immunogenic antigen and the frequency of responders to this protein tended to increase in P. vivax patients with higher parasitemia. For both antigens, IgG antibody responses tended to be lower in patients who had experienced their first bout of malaria. Furthermore, anemic patients presented higher IgG antibody responses to PvMSP-3 alpha(359-798). Since the humoral response involves a number of antibodies acting simultaneously on different targets, we performed a Principal Component Analysis (PCA). Anemic patients had, on average, higher first principal component scores (IgG1/IgG2/IgG3/IgG4 anti-MSP3 alpha), which were negatively correlated with hemoglobin levels. Since antibodies against PfMSP-3 have been strongly associated with clinical protection, we cannot exclude the possibility of a dual role of PvMSP-3 specific antibodies in both immunity and pathogenesis of vivax malaria. Our results confirm the high immunogenicity of the conserved C terminus of PvMSP-1 and points to the considerable immunogenicity of polymorphic PvMSP-3 alpha(359-798) during natural infection. (C) 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Functional analysis of Plasmodium falciparum merozoite antigens : implications for erythrocyte invasion and vaccine development

Malaria is a major human health problem and is responsible for over 2 million deaths per year. It is caused by a number of species of the genus Plasmodium, and Plasmodium falciparum is the causative agent of the most lethal form. Consequently, the development of a vaccine against this parasite is a priority. There are a number of stages of the parasite life cycle that are being targeted for the development of vaccines. Important candidate antigens include proteins on the surface of the asexual merozoite stage, the form that invades the host erythrocyte. The development of methods to manipulate the genome of Plasmodium species has enabled the construction of gain-of-function and loss-of-function mutants and provided new strategies to analyse the role of parasite proteins. This has provided new information on the role of merozoite antigens in erythrocyte invasion and also allows new approaches to address their potential as vaccine candidates.