The Synthetic Plasmodium falciparum Circumsporozoite Peptide PfCS102 as a Malaria Vaccine Candidate: A Randomized Controlled Phase I Trial

Background

Fully efficient vaccines against malaria pre-erythrocytic stage are still lacking. The objective of this dose/adjuvant-finding study was to evaluate the safety, reactogenicity and immunogenicity of a vaccine candidate based on a peptide spanning the C-terminal region of Plasmodium falciparum circumsporozoite protein (PfCS102) in malaria naive adults.

Methodology and Principal Findings

Thirty-six healthy malaria-naive adults were randomly distributed into three dose blocks (10, 30 and 100 µg) and vaccinated with PfCS102 in combination with either Montanide ISA 720 or GSK proprietary Adjuvant System AS02A at days 0, 60, and 180. Primary end-point (safety and reactogenicity) was based on the frequency of adverse events (AE) and of abnormal biological safety tests; secondary-end point (immunogenicity) on P. falciparum specific cell-mediated immunity and antibody response before and after immunization. The two adjuvant formulations were well tolerated and their safety profile was good. Most AEs were local and, when systemic, involved mainly fatigue and headache. Half the volunteers in AS02A groups experienced severe AEs (mainly erythema). After the third injection, 34 of 35 volunteers developed anti-PfCS102 and anti-sporozoite antibodies, and 28 of 35 demonstrated T-cell proliferative responses and IFN-γ production. Five of 22 HLA-A2 and HLA-A3 volunteers displayed PfCS102 specific IFN-γ secreting CD8⁺ T cell responses. Responses were only marginally boosted after the 3^rd vaccination and remained stable for 6 months. For both adjuvants, the dose of 10 µg was less immunogenic in comparison to 30 and 100 µg that induced similar responses. AS02A formulations with 30 µg or 100 µg PfCS102 induced about 10-folds higher antibody and IFN-γ responses than Montanide formulations.

Conclusions/Significance

PfCS102 peptide was safe and highly immunogenic, allowing the design of more advanced trials to test its potential for protection. Two or three immunizations with a dose of 30 µg formulated with AS02A appeared the most appropriate choice for such studies.

Trial Registration

Swissmedic.ch 2002 DR 1227     

A Full-Length Plasmodium falciparum Recombinant Circumsporozoite Protein Expressed by Pseudomonas fluorescens Platform as a Malaria Vaccine Candidate

The circumsporozoite protein (CSP) of Plasmodium falciparum is a major surface protein, which forms a dense coat on the sporozoite''s surface. Preclinical research on CSP and clinical evaluation of a CSP fragment-based RTS, S/AS01 vaccine have demonstrated a modest degree of protection against P. falciparum, mediated in part by humoral immunity and in part by cell-mediated immunity. Given the partial protective efficacy of the RTS, S/AS01 vaccine in a recent Phase 3 trial, further improvement of CSP-based vaccines is crucial. In this report, we describe the preclinical development of a full-length, recombinant CSP (rCSP)-based vaccine candidate against P. falciparum malaria suitable for current Good Manufacturing Practice (cGMP) production. Utilizing a novel high-throughput Pseudomonas fluorescens expression platform, we demonstrated greater efficacy of full-length rCSP as compared to N-terminally truncated versions, rapidly down-selected a promising lead vaccine candidate, and developed a high-yield purification process to express immunologically active, intact antigen for clinical trial material production. The rCSP, when formulated with various adjuvants, induced antigen-specific antibody responses as measured by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay (IFA), as well as CD4+ T-cell responses as determined by ELISpot. The adjuvanted rCSP vaccine conferred protection in mice when challenged with transgenic P. berghei sporozoites containing the P. falciparum repeat region of CSP. Furthermore, heterologous prime/boost regimens with adjuvanted rCSP and an adenovirus type 35-vectored CSP (Ad35CS) showed modest improvements in eliciting CSP-specific T-cell responses and anti-malarial protection, depending on the order of vaccine delivery. Collectively, these data support the importance of further clinical development of adjuvanted rCSP, either as a stand-alone product or as one of the components in a heterologous prime/boost strategy, ultimately acting as an effective vaccine candidate for the mitigation of P. falciparum-induced malaria.     

Structure of the Plasmodium falciparum Circumsporozoite Protein, a Leading Malaria Vaccine Candidate

The Plasmodium falciparum circumsporozoite protein (CSP) is critical for sporozoite function and invasion of hepatocytes. Given its critical nature, a phase III human CSP malaria vaccine trial is ongoing. The CSP is composed of three regions as follows: an N terminus that binds heparin sulfate proteoglycans, a four amino acid repeat region (NANP), and a C terminus that contains a thrombospondin-like type I repeat (TSR) domain. Despite the importance of CSP, little is known about its structure. Therefore, recombinant forms of CSP were produced by expression in both Escherichia coli (Ec) and then refolded (EcCSP) or in the methylotrophic yeast Pichia pastoris (PpCSP) for structural analyses. To analyze the TSR domain of recombinant CSP, conformation-dependent monoclonal antibodies that recognized unfixed P. falciparum sporozoites and inhibited sporozoite invasion of HepG2 cells in vitro were identified. These monoclonal antibodies recognized all recombinant CSPs, indicating the recombinant CSPs contain a properly folded TSR domain structure. Characterization of both EcCSP and PpCSP by dynamic light scattering and velocity sedimentation demonstrated that both forms of CSP appeared as highly extended proteins (R_h 4.2 and 4.58 nm, respectively). Furthermore, high resolution atomic force microscopy revealed flexible, rod-like structures with a ribbon-like appearance. Using this information, we modeled the NANP repeat and TSR domain of CSP. Consistent with the biochemical and biophysical results, the repeat region formed a rod-like structure about 21–25 nm in length and 1.5 nm in width. Thus native CSP appears as a glycosylphosphatidylinositol-anchored, flexible rod-like protein on the sporozoite surface.Malaria caused by Plasmodium falciparum is a serious global health issue, resulting in an estimated 1.5 million deaths annually, primarily among infants and young children. Ongoing multifaceted global intervention strategies to control malaria include drug treatment, insecticide usage, bed-net use, and vaccine development. However, parasite and mosquito control measures have met with limited success resulting from an increased drug and insecticide resistance within the Plasmodia and mosquito populations, respectively. Vaccine development represents an encouraging approach given that previous animal and human studies using irradiated sporozoites demonstrated the feasibility of producing an efficacious vaccine (1–3). Although the exact immunologic correlates of protection remain elusive, an abundance of evidence indicates that protection against liver stage parasites is complex, involving multiple immune mechanisms (4–11).To date, the majority of the pre-erythrocytic stage vaccine development has focused on the circumsporozoite protein (CSP),² the predominant surface antigen on sporozoites. CSP can be segmented into three regions as follows: the N-terminal region containing region I; the central repeat region; and the C-terminal region containing the thrombospondin-like type I repeat (TSR). Initial CSP vaccine development focused on the central repeat region that contains the immunodominant B cell epitope (12). However, vaccine constructs quickly evolved to incorporate both the central repeat region containing the B cell epitopes and the C terminus containing the TSR domain, T cell epitopes, and B cell epitopes (13, 14). Currently, the most advanced and moderately effective malaria vaccine, RTS,S, is composed of a portion of the central repeat and the C-terminal regions linked to the hepatitis B surface antigen (15). However, recent studies have highlighted the physiological importance of the N-terminal region (16–19). Rathore et al. (19) not only demonstrated the role of the N-terminal region in liver cell attachment but also identified along with Ancsin and Kisilevsky (16) an epitope within the N-terminal region that interacted with liver cells through heparin sulfate (18). Moreover, this epitope was not only found to be immunogenic but the resulting antibodies were determined to be inhibitory in a sporozoite invasion assay (18). Peptides corresponding to the N-terminal region (PpCS-(22–110) and PpCS-(65–110)) were also recognized by sera obtained from individuals living in malaria-endemic regions (17).To better understand the structure of CSP and to produce good quality recombinant protein for human vaccine-directed studies, we generated full-length and near full-length recombinant CSP. We examined two expression systems, Escherichia coli and Pichia pastoris, to determine their feasibility to generate CSP. To assist the characterization of the rCSPs, we generated a panel of monoclonal antibodies (mAbs) that were characterized biologically prior to being used to examine the rCSPs. Additionally, each of the rCSP molecules was extensively biochemically and biophysically characterized. The results collated together have enabled the molecular modeling of CSP as a long flexible, rod-like protein.     

Changing Malaria Epidemiology and Diagnostic Criteria for Plasmodium falciparum Clinical Malaria

Background

In tropical Africa, where malaria is highly endemic, low grade infections are asymptomatic and the diagnosis of clinical malaria is usually based on parasite density. Here we investigate how changes in malaria control and endemicity modify diagnostic criteria of Plasmodium falciparum attacks.

Methods and Findings

Parasitological and clinical data from the population of Dielmo, Senegal, monitored during 20 years, are analyzed in a random-effect logistic regression model to investigate the relationship between the level of parasitemia and risk of fever. Between 1990 and 2010, P. falciparum prevalence in asymptomatic persons declined from 85% to 1% in children 0–3 years and from 34% to 2% in adults ≥50 years. Thresholds levels of parasitemia for attributing fever episodes to malaria decreased by steps in relation to control policies. Using baseline threshold during following periods underestimated P. falciparum attacks by 9.8–20.2% in children and 18.9–40.2% in adults. Considering all fever episodes associated with malaria parasites as clinical attacks overestimated P. falciparum attacks by 42.2–68.5% in children and 45.9–211.7% in adults.

Conclusions

Malaria control modifies in all age-groups the threshold levels of parasitemia to be used for the assessment of malaria morbidity and to guide therapeutic decisions. Even under declining levels of malaria endemicity, the parasite density method must remain the reference method for distinguishing malaria from other causes of fever and assessing trends in the burden of malaria.     

Phase 1/2a Trial of Plasmodium vivax Malaria Vaccine Candidate VMP001/AS01B in Malaria-Naive Adults: Safety,Immunogenicity, and Efficacy

Background

A vaccine to prevent infection and disease caused by Plasmodium vivax is needed both to reduce the morbidity caused by this parasite and as a key component in efforts to eradicate malaria worldwide. Vivax malaria protein 1 (VMP001), a novel chimeric protein that incorporates the amino- and carboxy- terminal regions of the circumsporozoite protein (CSP) and a truncated repeat region that contains repeat sequences from both the VK210 (type 1) and the VK247 (type 2) parasites, was developed as a vaccine candidate for global use.

Methods

We conducted a first-in-human Phase 1 dose escalation vaccine study with controlled human malaria infection (CHMI) of VMP001 formulated in the GSK Adjuvant System AS01_B. A total of 30 volunteers divided into 3 groups (10 per group) were given 3 intramuscular injections of 15μg, 30μg, or 60μg respectively of VMP001, all formulated in 500μL of AS01_B at each immunization. All vaccinated volunteers participated in a P. vivax CHMI 14 days following the third immunization. Six non-vaccinated subjects served as infectivity controls.

Results

The vaccine was shown to be well tolerated and immunogenic. All volunteers generated robust humoral and cellular immune responses to the vaccine antigen. Vaccination did not induce sterile protection; however, a small but significant delay in time to parasitemia was seen in 59% of vaccinated subjects compared to the control group. An association was identified between levels of anti-type 1 repeat antibodies and prepatent period.

Significance

This trial was the first to assess the efficacy of a P. vivax CSP vaccine candidate by CHMI. The association of type 1 repeat-specific antibody responses with delay in the prepatency period suggests that augmenting the immune responses to this domain may improve strain-specific vaccine efficacy. The availability of a P. vivax CHMI model will accelerate the process of P. vivax vaccine development, allowing better selection of candidate vaccines for advancement to field trials.     

Improvement in Yield and Purity of a Recombinant Malaria Vaccine Candidate Based on the Receptor-Binding Domain of Plasmodium vivax Duffy Binding Protein by Codon Optimization

A recombinant blood-stage vaccine for Plasmodium vivax malaria based on the functional receptor-binding domain of PvDBP (PvRII) has been developed. A synthetic gene coding for PvRII was expressed in Escherichia coli using codon optimization. Expression level of recombinant PvRII was 10% of the total cellular proteins. Truncated PvRII products, seen when the native PvRII gene was expressed, were absent in case of synthetic gene.     

The structure of the Plasmodium falciparum EBA175 ligand domain and the molecular basis of host specificity

Erythrocyte-binding antigen 175 (EBA175) is one of the best-characterized Plasmodium falciparum merozoite ligands; the recently solved crystal structure of EBA175 reveals that terminal sialic acids on the erythrocyte glycoprotein glycophorin A are a crucial factor for erythrocyte recognition by EBA175 because they lock into pockets on its surface. Comparison with Plasmodium reichenowi EBA175 indicates that these interactions have a pivotal role in the host-specific adaptations of parasite ligands.     

A Phase I Randomized Clinical Trial of Candidate Human Immunodeficiency Virus type 1 Vaccine MVA.HIVA Administered to Gambian Infants

Background

A vaccine to decrease transmission of human immunodeficiency virus type 1 (HIV-1) during breast-feeding would complement efforts to eliminate infant HIV-1 infection by antiretroviral therapy. Relative to adults, infants have distinct immune development, potentially high-risk of transmission when exposed to HIV-1 and rapid progression to AIDS when infected. To date, there have been only three published HIV-1 vaccine trials in infants.

Trial Design

We conducted a randomized phase I clinical trial PedVacc 001 assessing the feasibility, safety and immunogenicity of a single dose of candidate vaccine MVA.HIVA administered intramuscularly to 20-week-old infants born to HIV-1-negative mothers in The Gambia.

Methods

Infants were followed to 9 months of age with assessment of safety, immunogenicity and interference with Expanded Program on Immunization (EPI) vaccines. The trial is the first stage of developing more complex prime-boost vaccination strategies against breast milk transmission of HIV-1.

Results

From March to October 2010, 48 infants (24 vaccine and 24 no-treatment) were enrolled with 100% retention. The MVA.HIVA vaccine was safe with no difference in adverse events between vaccinees and untreated infants. Two vaccine recipients (9%) and no controls had positive ex vivo interferon-γ ELISPOT assay responses. Antibody levels elicited to the EPI vaccines, which included diphtheria, tetanus, whole-cell pertussis, hepatitis B virus, Haemophilus influenzae type b and oral poliovirus, reached protective levels for the vast majority and were similar between the two arms.

Conclusions

A single low-dose of MVA.HIVA administered to 20-week-old infants in The Gambia was found to be safe and without interference with the induction of protective antibody levels by EPI vaccines, but did not alone induce sufficient HIV-1-specific responses. These data support the use of MVA carrying other transgenes as a boosting vector within more complex prime-boost vaccine strategies against transmission of HIV-1 and/or other infections in this age group.

Trial Registration

ClinicalTrials.gov NCT00982579 The Pan African Clinical Trials Registry PACTR2008120000904116     

Inhibition of the SR Protein-Phosphorylating CLK Kinases of Plasmodium falciparum Impairs Blood Stage Replication and Malaria Transmission

Cyclin-dependent kinase-like kinases (CLKs) are dual specificity protein kinases that phosphorylate Serine/Arginine-rich (SR) proteins involved in pre-mRNA processing. Four CLKs, termed PfCLK-1-4, can be identified in the human malaria parasite Plasmodium falciparum, which show homology with the yeast SR protein kinase Sky1p. The four PfCLKs are present in the nucleus and cytoplasm of the asexual blood stages and of gametocytes, sexual precursor cells crucial for malaria parasite transmission from humans to mosquitoes. We identified three plasmodial SR proteins, PfSRSF12, PfSFRS4 and PfSF-1, which are predominantly present in the nucleus of blood stage trophozoites, PfSRSF12 and PfSF-1 are further detectable in the nucleus of gametocytes. We found that recombinantly expressed SR proteins comprising the Arginine/Serine (RS)-rich domains were phosphorylated by the four PfCLKs in in vitro kinase assays, while a recombinant PfSF-1 peptide lacking the RS-rich domain was not phosphorylated. Since it was hitherto not possible to knock-out the pfclk genes by conventional gene disruption, we aimed at chemical knock-outs for phenotype analysis. We identified five human CLK inhibitors, belonging to the oxo-β-carbolines and aminopyrimidines, as well as the antiseptic chlorhexidine as PfCLK-targeting compounds. The six inhibitors block P. falciparum blood stage replication in the low micromolar to nanomolar range by preventing the trophozoite-to-schizont transformation. In addition, the inhibitors impair gametocyte maturation and gametogenesis in in vitro assays. The combined data show that the four PfCLKs are involved in phosphorylation of SR proteins with essential functions for the blood and sexual stages of the malaria parasite, thus pointing to the kinases as promising targets for antimalarial and transmission blocking drugs.     

Immunogenicity of a Recombinant Measles-HIV-1 Clade B Candidate Vaccine

Live attenuated measles virus is one of the most efficient and safest vaccines available, making it an attractive candidate vector for a HIV/AIDS vaccine aimed at eliciting cell-mediated immune responses (CMI). Here we have characterized the potency of CMI responses generated in mice and non-human primates after intramuscular immunisation with a candidate recombinant measles vaccine carrying an HIV-1 insert encoding Clade B Gag, RT and Nef (MV1-F4). Eight Mauritian derived, MHC-typed cynomolgus macaques were immunised with 10⁵ TCID₅₀ of MV1-F4, four of which were boosted 28 days later with the same vaccine. F4 and measles virus (MV)-specific cytokine producing T cell responses were detected in 6 and 7 out of 8 vaccinees, respectively. Vaccinees with either M6 or recombinant MHC haplotypes demonstrated the strongest cytokine responses to F4 peptides. Polyfunctional analysis revealed a pattern of TNFα and IL-2 responses by CD4+ T cells and TNFα and IFNγ responses by CD8+ T cells to F4 peptides. HIV-specific CD4+ and CD8+ T cells expressing cytokines waned in peripheral blood lymphocytes by day 84, but CD8+ T cell responses to F4 peptides could still be detected in lymphoid tissues more than 3 months after vaccination. Anti-F4 and anti-MV antibody responses were detected in 6 and 8 out of 8 vaccinees, respectively. Titres of anti-F4 and MV antibodies were boosted in vaccinees that received a second immunisation. MV1-F4 carrying HIV-1 Clade B inserts induces robust boostable immunity in non-human primates. These results support further exploration of the MV1-F4 vector modality in vaccination strategies that may limit HIV-1 infectivity.     

Defining the Antigenic Diversity of Plasmodium falciparum Apical Membrane Antigen 1 and the Requirements for a Multi-Allele Vaccine against Malaria

Apical Membrane Antigen 1 (AMA1) is a leading malaria vaccine candidate and a target of naturally-acquired human immunity. Plasmodium falciparum AMA1 is polymorphic and in vaccine trials it induces strain-specific protection. This antigenic diversity is a major roadblock to development of AMA1 as a malaria vaccine and understanding how to overcome it is essential. To assess how AMA1 antigenic diversity limits cross-strain growth inhibition, we assembled a panel of 18 different P. falciparum isolates which are broadly representative of global AMA1 sequence diversity. Antibodies raised against four well studied AMA1 alleles (W2Mef, 3D7, HB3 and FVO) were tested for growth inhibition of the 18 different P. falciparum isolates in growth inhibition assays (GIA). All antibodies demonstrated substantial cross-inhibitory activity against different isolates and a mixture of the four different AMA1 antibodies inhibited all 18 isolates tested, suggesting significant antigenic overlap between AMA1 alleles and limited antigenic diversity of AMA1. Cross-strain inhibition by antibodies was only moderately and inconsistently correlated with the level of sequence diversity between AMA1 alleles, suggesting that sequence differences are not a strong predictor of antigenic differences or the cross-inhibitory activity of anti-allele antibodies. The importance of the highly polymorphic C1-L region for inhibitory antibodies and potential vaccine escape was assessed by generating novel transgenic P. falciparum lines for testing in GIA. While the polymorphic C1-L epitope was identified as a significant target of some growth-inhibitory antibodies, these antibodies only constituted a minor proportion of the total inhibitory antibody repertoire, suggesting that the antigenic diversity of inhibitory epitopes is limited. Our findings support the concept that a multi-allele AMA1 vaccine would give broad coverage against the diversity of AMA1 alleles and establish new tools to define polymorphisms important for vaccine escape.     

Proteomic Analysis of Detergent-resistant Membrane Microdomains in Trophozoite Blood Stage of the Human Malaria Parasite Plasmodium falciparum

Intracellular pathogens contribute to a significant proportion of infectious diseases worldwide. The successful strategy of evading the immune system by hiding inside host cells is common to all the microorganism classes, which exploit membrane microdomains, enriched in cholesterol and sphingolipids, to invade and colonize the host cell. These assemblies, with distinct biochemical properties, can be isolated by means of flotation in sucrose density gradient centrifugation because they are insoluble in nonionic detergents at low temperature. We analyzed the protein and lipid contents of detergent-resistant membranes from erythrocytes infected by Plasmodium falciparum, the most deadly human malaria parasite. Proteins associated with membrane microdomains of trophic parasite blood stages (trophozoites) include an abundance of chaperones, molecules involved in vesicular trafficking, and enzymes implicated in host hemoglobin degradation. About 60% of the identified proteins contain a predicted localization signal suggesting a role of membrane microdomains in protein sorting/trafficking.To validate our proteomic data, we raised antibodies against six Plasmodium proteins not characterized previously. All the selected candidates were recovered in floating low-density fractions after density gradient centrifugation. The analyzed proteins localized either to internal organelles, such as the mitochondrion and the endoplasmic reticulum, or to exported membrane structures, the parasitophorous vacuole membrane and Maurer''s clefts, implicated in targeting parasite proteins to the host erythrocyte cytosol or surface. The relative abundance of cholesterol and phospholipid species varies in gradient fractions containing detergent-resistant membranes, suggesting heterogeneity in the lipid composition of the isolated microdomain population. This study is the first report showing the presence of cholesterol-rich microdomains with distinct properties and subcellular localization in trophic stages of Plasmodium falciparum.Plasmodium falciparum, the most deadly agent of human malaria, caused around 216 million infections and 655,000 deaths in 2010. The complex parasite life cycle involves the development in a mosquito vector of the Anopheles genus and eventual migration to a human host. In this host, asymptomatic multiplication in the liver cells is followed by parasite release into the bloodstream and erythrocyte invasion. Inside the erythrocytes, parasites grow (trophozoite stage) and multiply asexually (schizont stage), developing into highly specialized invasive forms (merozoites). A fraction of parasites differentiate into gametocytes, the gamete precursors necessary to complete the transmission cycle. Parasite blood stages, responsible for malaria pathogenesis and transmission, actively remodel the host erythrocyte, generating novel membrane compartments to sustain the export and sorting of proteins to the host cell cytosol, membrane skeleton, and plasma membrane. The parasitophorous vacuole membrane (PVM),¹ which surrounds the parasite throughout the erythrocytic cycle, is the site where exported proteins are translocated into the erythrocyte cytosol (1, 2). Membrane-bound structures of parasite origin, the so-called Maurer''s clefts (MCs) (3, 4), form functionally independent compartments at the red blood cell (RBC) periphery and mediate the sorting/assembly of virulence factors en route to the host cell surface (5). In addition, populations of different vesicles (25 and 80 nm) were identified in the RBC cytosol, suggesting the presence of vesicular mediated trafficking for the delivery of cargo to different destinations (6).Membranes are important sites for cellular signaling events, and many proteins with therapeutic potential localize in these cellular compartments (7, 8). Membrane microdomains enriched in sphingolipids and cholesterol, also referred to as lipid rafts, have been extensively studied in different cell types and gained particular interest for their roles in infection and pathogenesis (8, 9). These assemblies are small and dynamic and can be stabilized to form larger microdomains implicated in a wide range of fundamental cellular processes, which vary depending on cell type (10). Sphingolipids exhibit strong lateral cohesion, generating tightly packed regions in the membrane bilayer, and cholesterol acts as a spacer present in both membrane leaflets generating stable, liquid-ordered phase domains in the membrane bilayer (11). Distinct biochemical properties render these membrane assemblies insoluble in nonionic detergents at low temperature, allowing for their enrichment as detergent-resistant membranes (DRMs). Proteins with DRM-raft affinity include glycosylphosphatidyl inositol (GPI)-anchored proteins and acylated, myristoylated, and palmitoylated proteins (11). DRM rafts also restrict free diffusion of membrane proteins, thereby directing the trafficking of proteins and lipids to and from cellular compartments. Because of their endocytic and receptor clustering capacity, an increasing number of pathogens, including Plasmodium falciparum, utilize them when interacting with their target cells for invasion (9, 12).Even though cholesterol-rich membrane microdomains are implicated in fundamental processes in the parasite life cycle, Plasmodium is unable to synthesize sterols and depends entirely on hosts for its cholesterol supply. During merozoite invasion, lipid and protein components of the erythrocyte rafts are selectively recruited and incorporated into the nascent PVM (13, 14). Plasmodium liver stages utilize cholesterol internalized by low-density lipoprotein and synthesized by hepatocytes (15).To shed light on the organization and dynamics of these assemblies during parasite development inside the infected cell, we identified and validated the DRM-raft proteome of the P. falciparum trophozoite/early schizont. Detected proteins only partially overlap with DRM components of the P. falciparum late schizonts (16, 17) or the mixed blood stages of the rodent malaria agent P. berghei (18). Immunolocalization of selected DRM-associated proteins indicated that these assemblies may reside in both exported compartments (PVM, MCs) and intracellular membranes/organelles. The analysis of DRM lipids suggested that distinct microdomains exist in the infected erythrocyte that differ in their relative abundance of cholesterol and phospholipids.     

Construction of Transgenic Plasmodium berghei as a Model for Evaluation of Blood-Stage Vaccine Candidate of Plasmodium falciparum Chimeric Protein 2.9

Background

The function of the 19 kDa C-terminal region of the merozoite surface protein 1 (MSP1-19) expressed by Plasmodium has been demonstrated to be conserved across distantly related Plasmodium species. The green fluorescent protein (GFP) is a reporter protein that has been widely used because it can be easily detected in living organisms by fluorescence microscopy and flow cytometry.

Methodology and Results

In this study, we used gene targeting to generate transgenic P. berghei (Pb) parasites (designated as PfMSP1-19Pb) that express the MSP1-19 of P. falciparum (Pf) and the GFP reporter protein simultaneously. The replacement of the PbMSP1-19 locus by PfMSP1-19 was verified by PCR and Southern analysis. The expression of the chimeric PbfMSP-1 and the GFP was verified by Western blot and fluorescence microscopy, respectively. Moreover, GFP-expressing transgenic parasites in blood stages can be readily differentiated from other blood cells using flow cytometry. A comparion of growth rates between wild-type and the PfMSP1-19Pb transgenic parasite indicated that the replacement of the MSP1-19 region and the expression of the GFP protein were not deleterious to the transgenic parasites. We used this transgenic mouse parasite as a murine model to evaluate the protective efficacy in vivo of specific IgG elicited by a PfCP-2.9 malaria vaccine that contains the PfMSP1-19. The BALB/c mice passively transferred with purified rabbit IgG to the PfCP-2.9 survived a lethal challenge of the PfMSP1-19Pb transgenic murine parasites, but not the wild-type P. berghei whereas the control mice passively transferred with purified IgG obtained from adjuvant only-immunized rabbits were vulnerable to both transgenic and wild-type infections.

Conclusions

We generated a transgenic P. berghei line that expresses PfMSP1-19 and the GFP reporter gene simultaneously. The availability of this parasite line provides a murine model to evaluate the protective efficacy in vivo of anti-MSP1-19 antibodies, including, potentially, those elicited by the PfCP-2.9 malaria vaccine in human volunteers.     

重组裂殖子表面蛋白质1特异性抗体有效抑制恶性疟原虫体外生长

42kD恶性疟原虫裂殖子表面蛋白质 1C末端片段 (MSP1 42 )是当今重要的疟疾疫苗候选抗原。为获得大量构象正确的MSP1 42重组蛋白进行疫苗有效性试验 ,在毕氏酵母系统中分泌表达了MSP1 42重组蛋白。通过与一组特异性识别构象表位的单抗反应 ,该重组蛋白在重要构象表位上与天然蛋白质一致。由该蛋白质诱生的抗体能有效地抑制恶性疟原虫的体外生长 ,这些结果为进一步开展MSP1 42重组蛋白疫苗有效性试验提供了基础     

Clinical Factors for Severity of Plasmodium falciparum Malaria in Hospitalized Adults in Thailand

Plasmodium falciparum is a major cause of severe malaria in Southeast Asia, however, there is limited information regarding clinical factors associated with the severity of falciparum malaria from this region. We performed a retrospective case-control study to compare clinical factors and outcomes between patients with severe and non-severe malaria, and to identify clinical factors associated with the requirement for intensive care unit (ICU) admission of patients with severe falciparum malaria among hospitalized adults in Southeast Asia. A total of 255 patients with falciparum malaria in the Hospital for Tropical Diseases in Bangkok, Thailand between 2006 and 2012 were included. We identified 104 patients with severe malaria (cases) and 151 patients with non-severe malaria (controls). Patients with falciparum malaria with following clinical and laboratory characteristics on admission (1) referrals, (2) no prior history of malaria, (3) body temperature of >38.5°C, (4) white blood cell counts >10×10⁹/µL, (5) presence of schizonts in peripheral blood smears, and (6) albumin concentrations of <3.5 g/dL, were more likely to develop severe malaria (P<0.05). Among patients with severe malaria, patients who met ≥3 of the 2010 WHO criteria had sensitivity of 79.2% and specificity of 81.8% for requiring ICU admission. Multivariate analysis identified the following as independent associated factors for severe malaria requiring ICU admission; (1) ethnicity of Thai [odds ratio (OR) = 3.601, 95% confidence interval (CI) = 1.011–12.822] or Myanmar [OR = 3.610, 95% CI = 1.138–11.445]; (2) referrals [OR = 3.571, 95% CI = 1.306–9.762]; (3) no prior history of malaria [OR = 5.887, 95% CI = 1.354–25.594]; and (4) albumin concentrations of <3.5 g/dL [OR = 7.200, 95% CI = 1.802–28.759]. Our findings are important for the clinical management of patients with malaria because it can help early identification of patients that could develop severe malaria and require ICU admission. Early identification and the timely initiation of appropriate treatments may well improve the outcomes and reduce the mortality of these patients.     

Broad Blockade Antibody Responses in Human Volunteers after Immunization with a Multivalent Norovirus VLP Candidate Vaccine: Immunological Analyses from a Phase I Clinical Trial

Background

Human noroviruses (NoVs) are the primary cause of acute gastroenteritis and are characterized by antigenic variation between genogroups and genotypes and antigenic drift of strains within the predominant GII.4 genotype. In the context of this diversity, an effective NoV vaccine must elicit broadly protective immunity. We used an antibody (Ab) binding blockade assay to measure the potential cross-strain protection provided by a multivalent NoV virus-like particle (VLP) candidate vaccine in human volunteers.

Methods and Findings

Sera from ten human volunteers immunized with a multivalent NoV VLP vaccine (genotypes GI.1/GII.4) were analyzed for IgG and Ab blockade of VLP interaction with carbohydrate ligand, a potential correlate of protective immunity to NoV infection and illness. Immunization resulted in rapid rises in IgG and blockade Ab titers against both vaccine components and additional VLPs representing diverse strains and genotypes not represented in the vaccine. Importantly, vaccination induced blockade Ab to two novel GII.4 strains not in circulation at the time of vaccination or sample collection. GII.4 cross-reactive blockade Ab titers were more potent than responses against non-GII.4 VLPs, suggesting that previous exposure history to this dominant circulating genotype may impact the vaccine Ab response. Further, antigenic cartography indicated that vaccination preferentially activated preexisting Ab responses to epitopes associated with GII.4.1997. Study interpretations may be limited by the relevance of the surrogate neutralization assay and the number of immunized participants evaluated.

Conclusions

Vaccination with a multivalent NoV VLP vaccine induces a broadly blocking Ab response to multiple epitopes within vaccine and non-vaccine NoV strains and to novel antigenic variants not yet circulating at the time of vaccination. These data reveal new information about complex NoV immune responses to both natural exposure and to vaccination, and support the potential feasibility of an efficacious multivalent NoV VLP vaccine for future use in human populations.

Trial Registration

ClinicalTrials.gov NCT01168401