Evaluation of two sexual-stage antigens as bivalent transmission-blocking vaccines in rodent malaria.

BACKGROUND: Transmission-blocking vaccine (TBV) is a promising strategy for malaria elimination. It is hypothesized that mixing or fusing two antigens targeting different stages of sexual development may provide higher transmission-blocking activity than these antigens used individually. METHODS: A chimeric protein composed of fragments of Pbg37 and PSOP25 was designed and expressed the recombinant protein in Escherichia coli Rosetta-gami B (DE3). After immunizing mice with individual recombinant proteins Pbg37 and PSOP25, mixed proteins (Pbg37+PSOP25), or the fusion protein (Pbg37-PSOP25), the antibody titers of individual sera were analyzed by ELISA. IFA and Western blot were performed to test the reactivity of the antisera with the native proteins in the parasite. The transmission-blocking activity of the different immunization schemes was assessed using in vitro and in vivo assays. RESULTS: When Pbg37 and PSOP25 were co-administered in a mixture or as a fusion protein, they elicited similar antibody responses in mice as single antigens without causing immunological interference with each other. Antibodies against the mixed or fused antigens recognized the target proteins in the gametocyte, gamete, zygote, and ookinete stages. The mixed proteins or the fusion protein induced antibodies with significantly stronger transmission-reducing activities in vitro and in vivo than individual antigens. CONCLUSIONS: There was no immunological interference between Pbg37 and PSOP25. The bivalent vaccines, which expand the portion of the sexual development during which the transmission-blocking antibodies act, produced significantly stronger transmission-reducing activities than single antigens. Altogether, these data provide the theoretical basis for the development of combination TBVs targeting different sexual stages.

Combining Monophosphoryl Lipid A (MPL), CpG Oligodeoxynucleotide (ODN), and QS-21 Adjuvants Induces Strong and Persistent Functional Antibodies and T Cell Responses against Cell-Traversal Protein for Ookinetes and Sporozoites (CelTOS) of Plasmodium falciparum in BALB/c Mice.

Plasmodium falciparum cell-traversal protein for ookinetes and sporozoites (PfCelTOS) is an advanced vaccine candidate that has a crucial role in the traversal of the malaria parasite in both mosquito and mammalian hosts. As recombinant purified proteins are normally poor immunogens, they require to be admixed with an adjuvant(s); therefore, the objective of the present study was to evaluate the capacity of different vaccine adjuvants, monophosphoryl lipid A (MPL), CpG, and Quillaja saponaria Molina fraction 21 (QS-21), alone or in combination (MCQ [MPL/CpG/QS-21]), to enhance the immunogenicity of Escherichia coli-expressed PfCelTOS in BALB/c mice. This goal was achieved by the assessment of anti-PfCelTOS IgG antibodies (level, titer, IgG isotype profile, avidity, and persistence) and extracellular Th1 cytokines using an enzyme-linked immunosorbent assay (ELISA) on postimmunized BALB/c mouse sera and PfCelTOS-stimulated splenocytes, respectively. Also, an assessment of the transmission-reducing activity (TRA) of anti-PfCelTOS obtained from different vaccine groups was carried out in female Anopheles stephensi mosquitoes by using a standard membrane feeding assay (SMFA). In comparison to PfCelTOS alone, administration of PfCelTOS with three distinct potent Th1 adjuvants in vaccine mouse groups showed enhancement and improvement of PfCelTOS immunogenicity that generated more bias toward a Th1 response with significantly enhanced titers and avidity of the anti-PfCelTOS responses that could impair ookinete development in A. stephensi However, immunization of mice with PfCelTOS with MCQ mixture adjuvants resulted in the highest levels of induction of antibody titers, avidity, and inhibitory antibodies in oocyst development (88%/26.7% reductions in intensity/prevalence) in A. stephensi It could be suggested that adjuvant combinations with different mechanisms stimulate better functional antibody responses than adjuvants individually against challenging diseases such as malaria.

Preclinical immunogenicity and safety of the cGMP-grade placental malaria vaccine PRIMVAC.

BACKGROUND: VAR2CSA is the lead antigen for developing a vaccine that would protect pregnant women against placental malaria. A multi-system feasibility study has identified E. coli as a suitable bacterial expression platform allowing the production of recombinant VAR2CSA-DBL1x-2x (PRIMVAC) to envisage a prompt transition to current Good Manufacturing Practice (cGMP) vaccine production. METHODS: Extensive process developments were undertaken to produce cGMP grade PRIMVAC to permit early phase clinical trials. PRIMVAC stability upon storage was assessed over up to 3 years. A broad toxicology investigation was carried out in rats allowing meanwhile the analysis of PRIMVAC immunogenicity. FINDINGS: We describe the successful cGMP production of 4. 65 g of PRIMVAC. PRIMVAC drug product was stable and potent for up to 3 years upon storage at -20 °C and showed an absence of toxicity in rats. PRIMVAC adjuvanted with Alhydrogel® or GLA-SE was able to generate antibodies able to recognize VAR2CSA expressed at the surface of erythrocytes infected with different strains. These antibodies also inhibit the interaction of the homologous NF54-CSA strain and to a lower extend of heterologous strains to CSA. INTERPRETATION: This work paved the way for the clinical development of an easily scalable low cost effective vaccine that could protect against placental malaria and prevent an estimated 10,000 maternal and 200,000 infant deaths annually. FUND: This work was supported by a grant from the Bundesministerium für Bildung und Forschung (BMBF), Germany through Kreditanstalt für Wiederaufbau (KfW) (Reference No: 202060457) and through funding from Irish Aid, Department of Foreign Affairs and Trade, Ireland.

First-in-human, Randomized, Double-blind Clinical Trial of Differentially Adjuvanted PAMVAC, A Vaccine Candidate to Prevent Pregnancy-associated Malaria.

BACKGROUND: Malaria in pregnancy has major impacts on mother and child health. To complement existing interventions, such as intermittent preventive treatment and use of impregnated bed nets, we developed a malaria vaccine candidate with the aim of reducing sequestration of asexual "blood-stage" parasites in the placenta, the major virulence mechanism. METHODS: The vaccine candidate PAMVAC is based on a recombinant fragment of VAR2CSA, the Plasmodium falciparum protein responsible for binding to the placenta via chondroitin sulfate A (CSA). Healthy, adult malaria-naive volunteers were immunized with 3 intramuscular injections of 20 µg (n = 9) or 50 µg (n = 27) PAMVAC, adjuvanted with Alhydrogel or glucopyranosyl lipid adjuvant in stable emulsion (GLA-SE) or in a liposomal formulation with QS21 (GLA-LSQ). Allocation was random and double blind. The vaccine was given every 4 weeks. Volunteers were observed for 6 months following last immunization. RESULTS: All PAMVAC formulations were safe and well tolerated. A total of 262 adverse events (AEs) occurred, 94 (10 grade 2 and 2 grade 3) at least possibly related to the vaccine. No serious AEs occurred. Distribution and severity of AEs were similar in all arms. PAMVAC was immunogenic in all participants. PAMVAC-specific antibody levels were highest with PAMVAC-GLA-SE. The antibodies inhibited binding of VAR2CSA expressing P. falciparum-infected erythrocytes to CSA in a standardized functional assay. CONCLUSIONS: PAMVAC formulated with Alhydrogel or GLA-based adjuvants was safe, well tolerated, and induced functionally active antibodies. Next, PAMVAC will be assessed in women before first pregnancies in an endemic area. CLINICAL TRIALS REGISTRATION: EudraCT 2015-001827-21; ClinicalTrials.gov NCT02647489.

Fusion to Tetrahymena thermophila granule lattice protein 1 confers solubility to sexual stage malaria antigens in Escherichia coli.

A transmission-blocking vaccine targeting the sexual stages of Plasmodium species could play a key role in eradicating malaria. Multiple studies have identified the P. falciparum proteins Pfs25 and Pfs48/45 as prime targets for transmission-blocking vaccines. Although significant advances have been made in recombinant expression of these antigens, they remain difficult to produce at large scale and lack strong immunogenicity as subunit antigens. We linked a self-assembling protein, granule lattice protein 1 (Grl1p), from the ciliated protozoan, Tetrahymena thermophila, to regions of the ectodomains of either Pfs25 or Pfs48/45. We found that resulting protein chimera could be produced in E. coli as nanoparticles that could be readily purified in soluble form. When produced in the E. coli SHuffle strain, fusion to Grl1p dramatically increased solubility of target antigens while at the same time directing the formation of particles with diameters centering on 38 and 25 nm depending on the antigen. In a number of instances, co-expression with chaperone proteins and induction at a lower temperature further increased expression and solubility. Based on Western blotting and ELISA analysis, Pfs25 and Pfs48/45 retained their transmission-blocking epitopes within E. coli-derived particles, and the particles themselves elicited strong antibody responses in rabbits when given with an aluminum-based adjuvant. Antibodies against Pfs25-containing nanoparticles blocked parasite transmission in standard membrane-feeding assays. In conclusion, fusion to Grl1p can act as a solubility enhancer for proteins with limited solubility while retaining correct folding, which may be useful for applications such as the production of vaccines and other biologics.

Cryopreservation-related loss of antigen-specific IFNγ producing CD4+ T-cells can skew immunogenicity data in vaccine trials: Lessons from a malaria vaccine trial substudy.

Ex vivo functional immunoassays such as ELISpot and intracellular cytokine staining (ICS) by flow cytometry are crucial tools in vaccine development both in the identification of novel immunogenic targets and in the immunological assessment of samples from clinical trials. Cryopreservation and subsequent thawing of PBMCs via validated processes has become a mainstay of clinical trials due to processing restrictions inherent in the disparate location and capacity of trial centres, and also in the need to standardize biological assays at central testing facilities. Logistical and financial requirement to batch process samples from multiple study timepoints are also key. We used ELISpot and ICS assays to assess antigen-specific immunogenicity in blood samples taken from subjects enrolled in a phase II malaria heterologous prime-boost vaccine trial and showed that the freeze thaw process can result in a 3-5-fold reduction of malaria antigen-specific IFNγ-producing CD3+CD4+ effector populations from PBMC samples taken post vaccination. We have also demonstrated that peptide responsive CD8+ T cells are relatively unaffected, as well as CD4+ T cell populations that do not produce IFNγ. These findings contribute to a growing body of data that could be consolidated and synthesised as guidelines for clinical trials with the aim of increasing the efficiency of vaccine development pipelines.

An expanding toolkit for preclinical pre-erythrocytic malaria vaccine development: bridging traditional mouse malaria models and human trials.

Malaria remains a significant public health burden with 214 million new infections and over 400,000 deaths in 2015. Elucidating relevant Plasmodium parasite biology can lead to the identification of novel ways to control and ultimately eliminate the parasite within geographic areas. Particularly, the development of an effective vaccine that targets the clinically silent pre-erythrocytic stages of infection would significantly augment existing malaria elimination tools by preventing both the onset of blood-stage infection/disease as well as spread of the parasite through mosquito transmission. In this Perspective, we discuss the role of small animal models in pre-erythrocytic stage vaccine development, highlighting how human liver-chimeric and human immune system mice are emerging as valuable components of these efforts.

Evaluation of the Impact of Codon Optimization and N-Linked Glycosylation on Functional Immunogenicity of Pfs25 DNA Vaccines Delivered by In Vivo Electroporation in Preclinical Studies in Mice.

Plasmodium falciparum sexual stage surface antigen Pfs25 is a well-established candidate for malaria transmission-blocking vaccine development. Immunization with DNA vaccines encoding Pfs25 has been shown to elicit potent antibody responses in mice and nonhuman primates. Studies aimed at further optimization have revealed improved immunogenicity through the application of in vivo electroporation and by using a heterologous prime-boost approach. The goal of the studies reported here was to systematically evaluate the impact of codon optimization, in vivo electroporation, and N-linked glycosylation on the immunogenicity of Pfs25 encoded by DNA vaccines. The results from this study demonstrate that while codon optimization and in vivo electroporation greatly improved functional immunogenicity of Pfs25 DNA vaccines, the presence or absence of N-linked glycosylation did not significantly impact vaccine efficacy. These findings suggest that N-glycosylation of Pfs25 encoded by DNA vaccines is not detrimental to overall transmission-blocking efficacy.

Artesunate versus chloroquine infection-treatment-vaccination defines stage-specific immune responses associated with prolonged sterile protection against both pre-erythrocytic and erythrocytic Plasmodium yoelii infection.

Sterile protection against malaria infection can be achieved through vaccination of mice and humans with whole Plasmodium spp. parasites. One such method, known as infection-treatment-vaccination (ITV), involves immunization with wild type sporozoites (spz) under drug coverage. In this work, we used the different effects of antimalarial drugs chloroquine (CQ) and artesunate (AS) on blood stage (BS) parasites to dissect the stage-specific immune responses in mice immunized with Plasmodium yoelii spz under either drug, as well as their ability to protect mice against challenge with spz or infected RBCs (iRBCs). Whereas CQ-ITV induced sterile protection against challenge with both spz and iRBCs, AS-ITV only induced sterile protection against spz challenge. Importantly, AS-ITV delayed the onset of BS infection, indicating that both regimens induced cross-stage immunity. Moreover, both CQ- and AS-ITV induced CD8(+) T cells in the liver that eliminated malaria-infected hepatocytes in vitro, as well as Abs that recognized pre-erythrocytic parasites. Sera from both groups of mice inhibited spz invasion of hepatocytes in vitro, but only CQ-ITV induced high levels of anti-BS Abs. Finally, passive transfer of sera from CQ-ITV-treated mice delayed the onset of erythrocytic infection in the majority of mice challenged with P. yoelii iRBCs. Besides constituting the first characterization, to our knowledge, of AS-ITV as a vaccination strategy, our data show that ITV strategies that lead to subtle differences in the persistence of parasites in the blood enable the characterization of the resulting immune responses, which will contribute to future research in vaccine design and malaria interventions.

Community perceptions of a malaria vaccine in the Kintampo districts of Ghana.

BACKGROUND: Malaria remains the leading cause of morbidity and mortality in sub-Saharan Africa despite tools currently available for its control. Making malaria vaccine available for routine use will be a major hallmark, but its acceptance by community members and health professionals within the health system could pose considerable challenge as has been found with the introduction of polio vaccinations in parts of West Africa. Some of these challenges may not be expected since decisions people make are many a time driven by a complex myriad of perceptions. This paper reports knowledge and perceptions of community members in the Kintampo area of Ghana where malaria vaccine trials have been ongoing as part of the drive for the first-ever licensed malaria vaccine in the near future. METHODS: Both qualitative and quantitative methods were used in the data collection processes. Women and men whose children were or were not involved in the malaria vaccine trial were invited to participate in focus group discussions (FGDs). Respondents, made up of heads of religious groupings in the study area, health care providers, traditional healers and traditional birth attendants, were also invited to participate in in-depth interviews (IDIs). A cross-sectional survey was conducted in communities where the malaria vaccine trial (Mal 047RTS,S) was carried out. In total, 12 FGDs, 15 IDIs and 466 household head interviews were conducted. RESULTS: Knowledge about vaccines was widespread among participants. Respondents would like their children to be vaccinated against all childhood illnesses including malaria. Knowledge of the long existing routine vaccines was relatively high among respondents compared to hepatitis B and Haemophilus influenza type B vaccines that were introduced more recently in 2002. There was no clear religious belief or sociocultural practice that will serve as a possible barrier to the acceptance of a malaria vaccine. CONCLUSION: With the assumption that a malaria vaccine will be as efficacious as other EPI vaccines, community members in Central Ghana will accept and prefer malaria vaccine to malaria drugs as a malaria control tool. Beliefs and cultural practices as barriers to the acceptance of malaria vaccine were virtually unknown in the communities surveyed.

On designing stable magnetic vectors as carriers for malaria DNA vaccine.

Superparamagnetic iron oxide nanoparticles (SPIONs) can be used as therapeutic and diagnostic agents due to their unique magnetic characteristics, provided that they are stable in physiological conditions. Here, the assembly of different magnetic vector configurations comprising SPIONs, polyethylenimine (PEI), and hyaluronic acid (HA), acting as carriers for malaria DNA vaccine encoding Plasmodium yoelii merozoite surface protein MSP1-19 (VR1020-PyMSP1-19), and their stability in different cell media were investigated. The order of assembly affected vector size, surface charge, stability, and ability to bind and release DNA. Generally, all vectors showed relatively small size of less than 200 nm in water, whereas higher degree of aggregation was observed immediately after transferring to high-ionic strength media such as 150 mM NaCl buffer and RPMI 1640 culture media (Roswell Park Memorial Institute medium). However, the pre-addition of HA to DNA effectively reduced the extent of aggregation in serum-free RPMI 1640 with sizes of almost all complexes remaining below 90 nm, particularly at HA:PEI charge ratio of 100%. The presence of fetal bovine serum (FBS) in RPMI 1640 culture media further converted the surface charge of vectors from positive to negative, decreasing the size to smaller than 50 nm. Partial disassembly of some vectors was observed in water, in RPMI, and in RPMI supplemented with 10% FBS after incubation for 1h, but not in NaCl buffer, indicating that incubation of complexes in NaCl buffer prior to transfection may limit the intracellular release of plasmid DNA. DNase sensitivity assay showed that plasmid DNA vaccine encoding the PyMSP1-19 in all configurations preserved their structural integrity without damage, even after DNase I treatment for 30 min. This study demonstrated that structurally well-defined magnetic gene carriers could be designed to improve malaria DNA vaccine delivery systems, particularly for in vivo applications.

Immunomodulatory and physical effects of phospholipid composition in vaccine adjuvant emulsions.

Egg phosphatidylcholine is commonly used as an emulsifier in formulations administered parenterally. However, synthetic phosphatidylcholine (PC) emulsifiers are now widely available and may be desirable substitutes for egg-derived phospholipids due to stability, purity, and material source considerations. In earlier work, we demonstrated that a squalene-1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) emulsion provided equivalent physical stability compared to a squalene-egg PC emulsion. In the present manuscript, we evaluate the physical stability of vaccine adjuvant emulsions containing a range of other synthetic phosphatidylcholine emulsifiers. Besides the POPC emulsion, the 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) emulsion showed good particle size and visual stability compared to emulsions made with other synthetic phospholipids. Moreover, comparable immune responses were elicited by squalene emulsions employing various synthetic PC or egg PC emulsifiers in combination with an inactivated influenza vaccine or a recombinant malaria antigen, and these responses were generally enhanced compared to antigen without adjuvant. Therefore, we show that (1) some synthetic PCs (DMPC, POPC, and to a lesser extent 1,2-dioleoyl-sn-glycero-3-phosphocholine) are effective stabilizers of squalene emulsion over a range of storage temperatures while others are not (1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, and 1,2-dilauroyl-sn-glycero-3-phosphocholine) and (2) the immunogenicity of stable squalene emulsions is similar regardless of PC source.

The effect of adjuvants on the immune response induced by a DBL4ɛ-ID4 VAR2CSA based Plasmodium falciparum vaccine against placental malaria.

A vaccine protecting women against placental malaria could be based on the sub-domains of the VAR2CSA antigen, since antibodies against the DBL4ɛ-ID4 subunit of the VAR2CSA protein can inhibit parasite binding to the placental ligand chondroitin sulphate A (CSA). Here we tested the ability of DBL4ɛ-ID4 to induce binding-inhibitory antibodies when formulated with adjuvants approved for human use. We have characterized the immune response of DBL4ɛ-ID4 in combination with Freund's complete and incomplete adjuvant and with three adjuvants currently being used in clinical trials: Montanide(®) ISA 720, Alhydrogel(®) and CAF01. Antibodies induced against DBL4ɛ-ID4 in combination with these adjuvants inhibited parasite binding to CSA from 82% to 99%. Although, different epitope recognition patterns were obtained for the different formulations, all adjuvant combinations induced strong Th1 and Th2 type responses, resulting in IgG with similar binding strength, with to the DBL4ɛ-ID4 antigen. These results demonstrate that the DBL4ɛ-ID4 antigen is highly immunogenic and that binding inhibitory antibodies are induced when formulated with any of the tested adjuvants.

Prospects for malaria elimination in non-Amazonian regions of Latin America.

Latin America contributes 1-1.2 million clinical malaria cases to the global malaria burden of about 300 million per year. In 21 malaria endemic countries, the population at risk in this region represents less than 10% of the total population exposed worldwide. Factors such as rapid deforestation, inadequate agricultural practices, climate change, political instability, and both increasing parasite drug resistance and vector resistance to insecticides contribute to malaria transmission. Recently, several malaria endemic countries have experienced a significant reduction in numbers of malaria cases. This is most likely due to actions taken by National Malaria Control Programs (NMCP) with the support from international funding agencies. We describe here the research strategies and activities to be undertaken by the Centro Latino Americano de Investigación en Malaria (CLAIM), a new research center established for the non-Amazonian region of Latin America by the National Institute of Allergy and Infectious Diseases (NIAID). Throughout a network of countries in the region, initially including Colombia, Guatemala, Panama, and Peru, CLAIM will address major gaps in our understanding of changing malaria epidemiology, vector biology and control, and clinical malaria mainly due to Plasmodium vivax. In close partnership with NMCPs, CLAIM seeks to conduct research on how and why malaria is decreasing in many countries of the region as a basis for developing and implementing new strategies that will accelerate malaria elimination.

Enhancing immunogenicity to PLGA microparticulate systems by incorporation of alginate and RGD-modified alginate.

Poly-lactide-co-glycolide acid (PLGA) and alginate represent two different families of polymers widely used for microencapsulation application, even more, for vaccination purposes as particulate delivery/adjuvant systems. Combination of these polymers has been previously considered for tissue engineering and drug delivery, however there is currently no report regarding their combination for vaccine application. In the present work, a w/o/w solvent extraction technique was developed to prepare novel 1µm microparticles (MP) composed of PLGA and a small percentage of alginate (PLGA-alg MP). In addition, RGD-modified alginate was also employed as biofunctionalized material favoring MP-cell interaction (PLGA-alg-RGD MP). Two malaria synthetic peptides, SPf66 and S3, were microencapsulated into PLGA, PLGA-alg and PLGA-alg-RGD MP. The diverse MP formulations resulted very similar in terms of size and morphology, although the addition of alginate improved encapsulation efficiency and reduced the amount of surface adsorbed peptide. Immunization studies in Balb/c mice by intradermal route demonstrated that incorporation of alginate elicited higher humoral and cellular immune responses leading to more balanced Th1/Th2 responses. Furthermore, administration of MP containing RGD-modified alginate showed evidence of cell targeting by enhancing immunogenicity of microparticles, in particular with regard to cellular responses such as IFN-γ secretion and lymphoproliferation.

Long term stability of a recombinant Plasmodium falciparum AMA1 malaria vaccine adjuvanted with Montanide(®) ISA 720 and stabilized with glycine.

Plasmodium falciparum apical membrane antigen 1 (AMA1) is an asexual blood-stage vaccine candidate against the malaria parasite. AMA1-C1/ISA 720 refers to a mixture of recombinant AMA1 proteins representing the FVO and 3D7 alleles in 1:1 mass ratio, formulated with Montanide(®) ISA 720 as a water-in oil emulsion. In order to develop the AMA1-C1/ISA 720 vaccine for human use, it was important to determine the shelf life of this formulation. Previously it was found 267 mM glycine stabilized the proteins in Montanide(®) ISA 720 formulations for a short period of time at 2-8°C [25]. We now test the long term stability of AMA1-C1 at 10 and 40 µg/mL formulated with Montanide(®) ISA 720 with 50mM glycine as a stabilizer. Stability of AMA1-C1/ISA 720 at different time points following formulation (0, 5, 12 or 18 months) was evaluated by determining the mean particle size (diameter of the mean droplet volume), total protein content by a Modified Lowry assay, identity and integrity using western blot and SDS-PAGE. Our results showed that the mean particle size of these emulsions increased over time, whereas protein content, as determined by an ELISA method using a monoclonal antibody against penta-his, decreased over time. For the 10 µg/mL AMA1-C1/ISA 720 vaccine, the protein content was 6.5±2.2 µg/mL, and for the 40 µg/mL AMA1-C1/ISA 720 vaccine, the protein content was only 8.2±2.3 µg/mL after 18 months of storage at 2-8°C. These results suggest that the integrity of the protein was affected by long-term storage. The results of the present study indicate that the AMA1-C1/ISA 720 emulsion was unstable after 12 months of storage, after which AMA1-C1 proteins were partially degraded.

Safety and immunogenicity of a recombinant nonglycosylated erythrocyte binding antigen 175 Region II malaria vaccine in healthy adults living in an area where malaria is not endemic.

Erythrocyte binding antigen region II (EBA-175) is a conserved antigen of Plasmodium falciparum that is involved in binding of the parasite to the host's erythrocytes. We evaluated the safety and immunogenicity of a recombinant EBA-175 vaccine with aluminum phosphate adjuvant in healthy young adults living in the United States. Eighteen subjects/group received ascending doses (5, 20, 80, or 160 µg) of the vaccine at 0, 1, and 6 months; 8 subjects received placebo. Most of the injection site and systemic reactions were mild to moderate in intensity. After 2 or 3 doses of the vaccine at any concentration, antibody levels measured by enzyme-linked immunosorbent assay were significantly higher than those for the placebo group. Sera from subjects who received 3 doses of the vaccine at any concentration inhibited the growth of erythrocyte-stage P. falciparum at low levels compared to sera from placebo recipients or preimmune sera. In conclusion, the EBA-175 vaccine with adjuvant was safe and immunogenic in malaria-naïve subjects.

Evidences of protection against blood-stage infection of Plasmodium falciparum by the novel protein vaccine SE36.

An effective malaria vaccine is a public health priority. Proteins expressed during the blood-stage of the parasite life cycle have been proposed as good vaccine candidates. No such blood-stage vaccine, however, is available against Plasmodium falciparum, the deadliest Plasmodium species. We show here that P. falciparum serine repeat antigen 5 (SERA5) is a potential vaccine immunogen. We have constructed a new recombinant molecule of SERA5, namely SE36, based on previously reported SE47' molecule by removing the serine repeats. Epidemiological study in the holo-endemic population of Solomon Islands shows highly significant correlation of sero-conversion and malaria protective immunity against this antigen. Animal experiments using non-human primates, and a human phase 1a clinical trial assessed SE36 vaccine immunogenicity. Vaccination of squirrel monkeys with SE36 protein and aluminum hydroxyl gel (SE36/AHG) conferred protection against high parasitemia and boosted serum anti-SE36 IgG after P. falciparum parasite challenge. SE36/AHG was highly immunogenic in chimpanzees, where serum anti-SE36 IgG titers last more than one year. Phase 1a clinical trial (current controlled trials, ISRCTN78679862) demonstrated the safety and immunogenicity of SE36/AHG with 30 healthy adults and 10 placebo controls. Three subcutaneous administrations of 50 and 100microg dose of SE36/AHG were well-tolerated, with no severe adverse events; and resulted in 100% sero-conversion in both dose arms. The current research results for SE36/AHG provide initial clinical validation for future trials and suggest clues/strategies for further vaccine development.

Development of self-assembled nanoceramic carrier construct(s) for vaccine delivery.

Hydroxyapatite (HA) has been extensively investigated as scaffolds for tissue engineering, as drug delivery agents, as non-viral gene carriers, as prosthetic coatings, and composites. Recent studies in our laboratory demonstrated the immunoadjuvant properties of HA when administered with malarial merozoite surface protein-1(19) (MSP-1(19)). HA nanoceramic carrier was prepared by co-precipitation method that comprises of sintering and spray-drying technique. Prepared systems were characterized for crystallinity, size, shape, and antigen loading efficiency. Small size and large surface area of prepared HA demonstrated good adsorption efficiency of immunogens. Prepared nanoceramic formulations also showed slower in vitro antigen release and slower biodegrability behavior, which may lead to a prolonged exposure to antigen-presenting cells and lymphocytes. Furthermore, addition of mannose in nanoceramic formulation may additionally lead to increased stability and immunological reactions. Immunization with MSP-1(19) in nanoceramic-based adjuvant systems induced a vigorous immunoglobulin G (IgG) response, with higher IgG2a than IgG1 titers. In addition considerable amount of IFN-g and IL-2 was observed in spleen cells of mice immunized with nanoceramic-based vaccines. On the contrary, mice immunized with MSP-1(19) alone or with alum did not exhibit a significant cytotoxic response. The antibody responses to vaccine co-administered with HA was a mixed Th1/Th2 compared to the Th2-biased response obtained with alum. The prepared HA nanoparticles exhibit physicochemical properties that appear promising to make them a suitable immunoadjuvant to be used as antigen carriers for immunopotentiation.

Clinical trials to estimate the efficacy of preventive interventions against malaria in paediatric populations: a methodological review.

BACKGROUND: Recent years have seen publication of a considerable number of clinical trials of preventive interventions against clinical malaria in children. There has been variability in the specification of end-points, case definitions, analysis methods and reporting and the relative lack of standardization complicates the ability to make comparative evaluations between trials. METHODS: To prepare for a WHO consultation on design issues in malaria vaccine trials, controlled trials of preventive interventions against malaria in children in endemic countries were identified in which clinical malaria, or death, had been one of the main end-points. Trials were included that evaluated the impact of vaccines, insecticide-treated bed nets (ITN), intermittent presumptive or preventive therapy in infants (IPTi) or, in one instance, vitamin A supplementation. Methods that had been used in these trials were summarized and compared in order to identify issues that were directly relevant to the design of malaria vaccine trials. RESULTS: 29 controlled trials of preventive malaria interventions were identified, of which eight were vaccine trials. Vaccine trials that were designed to detect an effect on clinical malaria all reported the incidence rate of first episodes of clinical malaria as their primary endpoint. Only one trial of a preventive intervention (of ITN) was identified that was designed to detect an effect on severe malaria. A group of larger trials were designed to detect an effect of impregnated bed nets or curtains on all-cause mortality as the primary end-point. Key methodological and reporting differences between trials are noted in the text. Two issues have been identified that are of some concern. Firstly, the choice of primary endpoint is not stated in the reports of a number of the trials and, secondly, the relationship between pre-specified analysis plans and trial reports is rarely made clear. CONCLUSION: This article reports an investigation into the ways in which trial design and reporting could be improved and standardized to enable comparative evaluation of the relative merits of malaria control measures, and specifically with respect to the design of malaria vaccine trials. The need for standardization of clinical trial design, conduct, analysis and reporting has been also affirmed as a priority area by the Malaria Vaccine Technology Roadmap.